Identification of rnai targets and use of rnai for rational therapy of chemotherapy-resistant leukemia and other cancers

ABSTRACT

Provided is a mosaic mouse model for use in determining the potency of an shRNA in vivo for reducing survival of cancer cells of chemotherapy-resistant leukemia. The syngeneic mouse recipient is transplanted with tet-on competent leukemia cells carrying a bicistronic nucleic acid construct comprising a promoter operably linked to a fusion gene associated with chemotherapy-resistant leukemia, and a sequence encoding a reverse tet-transactivator protein, such that both coding sequences are co-expressed from the promoter. Also provided are methods of treating soft tissue cancers.

This application is a continuation of U.S. application Ser. No.13/260,540, filed Jun. 4, 2012, which was the U.S. National Stage ofInternational Application No. PCT/US2010/029083, filed on Mar. 29, 2010,which claims the benefit of the filing date of U.S. Provisional PatentApplication No. 61/164,125, filed Mar. 27, 2009, the contents of each ofwhich are hereby incorporated by reference in their entireties.

This patent disclosure contains material which is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosure,as it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

Throughout this application, patent applications, published patentapplications, issued and granted patents, texts, and literaturereferences are cited. For the purposes of the United States and otherjurisdictions that allow incorporation by reference, the disclosures ofthese publications are incorporated by reference into this application.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 20, 2013, isnamed 287000.157US2_SL.txt and is 73,601 bytes in size.

1. BACKGROUND OF THE INVENTION

This invention relates in part to the use of RNA interference (RNAi)technology in cancer cells to knock out genes encoding DNA replicationproteins, resulting in cell cycle arrest, and cytotoxicity. RNAitechnology enables specific suppression of the expression of virtuallyany gene and provides a new tool for drug target discovery, validation,and therapy. To obtain functional RNAi reagents for biological,biomedical, and clinical applications, it is important to identifypotent interfering RNA molecules (RNAi molecules) for a gene ofinterest.

Cancer is the second leading cause of death in industrialized countries,resulting from a combination of mutations in certain oncogenes and tumorsuppressor genes. Cancer can arise due to deregulation at many points ofthe cell cycle and during cell differentiation. Cell cycle checkpointsare a critical mechanism for assessing DNA damage. When damage is found,the checkpoint either arrests the cell cycle until the damage isrepaired or targets the cell for destruction via apoptosis if repairscannot be made.

Chromosomal translocations involving the MLL gene on chromosome 11q23are found in about 10% of all human leukemias and define one of the mostadverse genetic markers associated with chemotherapy resistance and poorprognosis. MLL is a H3K4 methyltransferase that acts as transcriptionalmaster regulator in normal hematopoiesis. Fusion proteins resulting from11q23 translocations are thought to misdirect transcriptionalactivation, reestablish self-renewal capacity and thereby promoteleukemogenesis. Various MLL cofactors, as well as downstream targetgenes, have been shown to be involved in MLL-leukemogenesis and havebeen proposed as targets for therapeutic intervention.

2. SUMMARY OF THE INVENTION

The role of particular DNA replication genes in cancer cellproliferation is not well understood. In the present inventiontet-regulated in vivo RNAi was applied to evaluate the role of candidatedrug target genes in established MLL-fusion leukemia. To facilitatethese studies a bioluminescent tet-on competent AML mouse model wasestablished based on cooperation of MLL/AF9 and oncogenic Nras, whichreflects a common genetic association in human AML. Coexpression ofMLL/AF9 and Nras^(G12D) resulted in aggressive myelomonocytic leukemias(mean survival 21 days), which are refractory to combined chemotherapy.To evaluate the potency of single-gene directed approaches in theseresistant AMLs a series of tet-regulatable shRNAs targeting (i)essential genes involved in DNA replication [Rpa1, Rpa3, PCNA], (ii) MLLassociated genes [MLL, HoxA9, Meis1, Men1, Dot1L, Myb], (iii) genesencoding epigenetic modifiers [AOF2, EED, HDAC, MEN1, SMARCA4, SMARCD1,SUZ12, WHSC 111] and (iii) other controls [Luciferase, Braf], weretransduced and the effects of doxycycline-induced shRNA expression wasstudied in vitro and in vivo using a dual-color tet-shRNA vector thatallows precise tracking of shRNA expressing cells.

In results of these experiments potent antileukemic effects of multipleshRNAs targeting Rpa1, Rpa3, PCNA and certain MLL associated genes (MLLitself, Meis1, Men1, Myb), and certain epigenetic modifier genes (Eed,Suz12, Aof2, Smarca4, Smarcd1, Men1, Hdac3, and Whs111), were seen. Inparallel assays using tet-on competent Mefs the antileukemic effects ofshRNAs targeting MLL-associated genes as well as certain epigeneticmodifier genes are shown not to be due to general cytotoxicity. Afterselection shRNA carrying MLL/AF9+Nras AML cells were transplanted intosecondary recipient mice. Following leukemia onset in bioluminescentimaging mice were treated with oral doxycycline, which induced rapid anddurable remissions upon induction of Rpa3 and Myb shRNAs, while thoseexpressing Braf control shRNAs progressed under therapy. Similar rapidand durable remissions were achieved using shRNAs targeting certainepigenetic modifier genes. These results have characterized a number ofgenes as essential for the survival of established MLL/AF9 induced AML,amongst them four MLL-associated genes (MLL, Men1, Meis1, Myb) that aredispensable in Mefs and seven epigenetic modifier genes (Eed, Suz12,Aof2, Smarca4, Smarcd1, Men1, Hdac3, and Whs111) that are dispensiblewhen tested in four non-transformed hematopoietic cell lines. Thesestudies demonstrate the power of tet-regulated RNAi to identify andevaluate genetic Achilles' heels in chemotherapy-resistant leukemia.

Using RNAi, we screened DNA replication genes in vitro for modifiers ofhuman cancer cell proliferation. The in vitro RNAi screens yieldedsurprising results: inhibitory RNAs targeting some DNA replication geneswere strongly anti-proliferative, while knockdown of many other suchgenes did not confer strong anti-proliferative effects. In particular,si- and shRNAs targeting genes encoding replication protein A3 (RPA3),ribonucleotide reductase M1 (RRM1), cell division cycle 45 (CDC45) andpescadillo 1 (PES1) significantly impair human cancer cellproliferation.

In vivo mouse leukemia models expressing inducible shRNAs were designedin order to identify particular RNAi molecules exhibiting the mostpotent in vivo efficacy for therapeutically curing chemoresistantleukemia, in particular leukemia involving MLL gene rearrangements. Inparticular, inducible shRNAs targeting genes encoding replicationprotein A3 (RPA3) and MYB exhibited potent efficacy in curingchemotherapy resistant leukemia in vivo, while numerous other inducibleshRNAs targeted against genes putatively involved in cancer cellproliferation had only marginal effects.

In one aspect, the invention provides a method for inhibitingproliferation of a cancer cell, the method comprising introducing intothe cancer cell a small interfering RNA (siRNA) comprising a sequencethat is complementary to a nucleotide sequence of a target gene encodinga DNA replication protein. In some embodiments, the method can furthercomprise administering a chemotherapy drug to the cancer cell to furtherinhibit proliferation of the cell. In some embodiments, cellproliferation can be inhibited through cell death (apoptosis). Cancercell proliferation can be inhibited in cultured cells in vitro, forexample in screening methods. Cancer cell proliferation can also beinhibited in vivo, in a therapeutic context. Chemotherapy drugs that canbe used according to the method include, but are not limited to, analkylating agent, a nitrosourea, an anti-metabolite, a topoisomeraseinhibitor, a mitotic inhibitor, an anthracycline, a corticosteroidhormone, a sex hormone, a targeted anti-tumor compound, and combinationsthereof.

In a certain embodiment of the invention, the DNA replication protein isselected from the group consisting of RPA3, RRM1, CDC45, and PES1. Insome embodiments, the siRNA can comprise a sequence selected from thefollowing:

RPA3: (SEQ ID NO: 1) CAUCUUAUGUCCAGUUUAA (SEQ ID NO: 2)CACCAUCUUGUGUACAUCU RRM1: (SEQ ID NO: 3) CAGAUCUUUGAAACUAUUU CDC45:(SEQ ID NO: 4) CAGUCAAUGUCGUCAAUGUAU PES1: (SEQ ID NO: 5)GCCUUGAGAAGAAGAAGUA (SEQ ID NO: 6) GUUGGACUCCGAGAGUUGU (SEQ ID NO: 7)CGGAACAAAGCCCGGAAGA

In another aspect, the invention provides a method for inhibitingproliferation of a cancer cell, the method comprising introducing intothe cancer cell an expression vector comprising a sequence encoding ashort hairpin RNA (shRNA) operably linked to a RNA polymerase promoter,wherein the shRNA comprises a loop and a duplex region that comprises asequence complementary to a nucleotide sequence of a target geneencoding a DNA replication protein. In some embodiments, the sequenceencoding the shRNA can be operably linked to an inducible promoter. Insome embodiments the inducible promoter can be a tet-responsive TREpromoter. In some embodiments, the method can further compriseadministering a chemotherapy drug to the cancer cell. Chemotherapy drugsthat can be used according to the method include, but are not limitedto, an alkylating agent, a nitrosourea, an anti-metabolite, atopoisomerase inhibitor, a mitotic inhibitor, an anthracycline, acorticosteroid hormone, a sex hormone, a targeted anti-tumor compound,and combinations thereof.

In a certain embodiment of the invention, the DNA replication protein isselected from the group consisting of RPA3, RRM1, CDC45 and PES1. Insome embodiments, the sequence encoding the shRNA can be or comprise asequence selected from the following:

RPA3: (SEQ ID NO: 8) TGCTGTTGACAGTGAGCGCACATCTTATGTCCAGTTTAAATAGTGAAGCCACAGATGTATTTAAACTGGACATAAGATGTATGCCTACTGCCTCGGA (SEQ ID NO: 9)TGCTGTTGACAGTGAGCGACCACCATCTTGTGTACATCTTTAGTGAAGCCACAGATGTAAAGATGTACACAAGATGGTGGCTGCCTACTGCCTCGGA RRM1: (SEQ ID NO: 10)TGCTGTTGACAGTGAGCGCGCAGATCTTTGAAACTATTTATAGTGAAGCCACAGATGTATAAATAGTTTCAAAGATCTGCTTGCCTACTGCCTCGGA CDC45: (SEQ ID NO: 11)TGCTGTTGACAGTGAGCGACCAGTCAATGTCGTCAATGTATAGTGAAGCCACAGATGTATACATTGACGACATTGACTGGCTGCCTACTGCCTCGGA PES1: (SEQ ID NO: 12)TGCTGTTGACAGTGAGCGCGGCCTTGAGAAGAAGAAGTATTAGTGAAGCCACAGATGTAATACTTCTTCTTCTCAAGGCCTTGCCTACTGCCTCGGA (SEQ ID NO: 13)TGCTGTTGACAGTGAGCGACGTTGGACTCCGAGAGTTGTATAGTGAAGCCACAGATGTATACAACTCTCGGAGTCCAACGCTGCCTACTGCCTCGGA (SEQ ID NO: 14)TGCTGTTGACAGTGAGCGACCGGAACAAAGCCCGGAAGAATAGTGAAGCCACAGATGTATTCTTCCGGGCTTTGTTCCGGGTGCCTACTGCCTCGGA

In some embodiments, the expression vector is a plasmid or a viralvector. In a certain embodiment, the viral vector is a retroviralvector, for example, a lentiviral vector. Where the vector is aretroviral vector, it can further comprise long terminal repeat (LTR)sequences located 5′ and/or 3′ to the sequence encoding the shRNA.

RNA polymerase promoters suitable for use in the invention include RNApolymerase II (pol II) and RNA polymerase III (pol III) promoters. Inone embodiment, the pol II promoter is selected from a CMV promoter anda U1 promoter. In another embodiment, the pol III promoter is selectedfrom a U6 promoter, an H1 promoter, and an SRP promoter. In someembodiments, the promoter is a U6 promoter. In some embodiments, thepromoter can be an inducible promoter. In some embodiments the induciblepromoter can be a tet-responsive TRE promoter.

Expression of the shRNA in the cell can be transient or stable. Incertain embodiments, the expression vector is episomal, resulting intransient expression of the shRNA. In other embodiments, the expressionvector is chromosomally integrated and produces a stably expressing cellline. In certain embodiments expression of the shRNA in the cell can beinducible. In certain embodiments expression of the shRNA in the cellcan be suppressable.

The shRNA comprises a stem region and a loop region. The stem regioncomprises a double-stranded (duplex) region of base paired nucleotides.The duplex region can comprise from 19 to 29 base pairs. The base pairscan be contiguous or non-contiguous. In a certain embodiment, the duplexregion contains 29 contiguous or non-contiguous base pairs. The loopregion is useful at 3 to 23 nucleotides in length.

In one aspect, the invention provides a method for identifying acompound that enhances the therapeutic efficacy of an RNAi molecule invivo for eliminating inhibits cell proliferation. The method cancomprise (1) introducing into a cancer cell an RNAi molecule that iscomplementary to a nucleotide sequence of a target gene, wherein thetarget gene encodes a DNA replication protein; (2) contacting the cancercell with a candidate compound; and (3) determining whether thecandidate compound inhibits cell proliferation, thereby identifying acompound that inhibits cell proliferation. In one embodiment, the methodfurther comprises (4) administering an effective amount of the compoundto a non-human animal having a tumor; and (5) monitoring tumor growth inthe non-human animal. In some embodiments, the method can furthercomprise comparing tumor growth in the non-human animal treated with thecompound to tumor growth in the non-human animal not treated with thecompound. The RNAi molecule introduced into a cancer cell can be a smallinterfering RNA (siRNA) or a short hairpin RNA (shRNA). In oneembodiment, the siRNA comprises a nucleic acid sequence that iscomplementary to a nucleotide sequence of a target gene, wherein thetarget gene encodes a DNA replication protein. In a further embodiment,the shRNA is operably linked to a RNA polymerase promoter, wherein theshRNA comprises a loop and a duplex region, wherein the duplex regioncomprises a sequence that is complementary to a nucleotide sequence of atarget gene, and wherein the target gene encodes a DNA replicationprotein. The DNA replication protein can be selected from the groupconsisting of replication protein A3 (RPA3), ribonucleotide reductase M1(RRM1), cell division cycle 45 (CDC45) and pescadillo 1 (PES1). In someembodiments, the non-human animal can be a mouse.

The invention provides a mosaic mouse model for chemotherapy-resistantleukemia. The model can be used to design rational cancer therapiesbased on the particular genotype of the cancer cells. For example, insome instances, particular gene fusions are responsible for conferringchemoresistance. Determination of such oncogenic genotypes canfacilitate treatment design. In particular, the vectors of the inventionprovide an enhanced screening system based on robust RNAi expression.RNAi molecules directed against the oncogene strongly and specificallydeplete the cancer cells.

The bicistronic vectors of the invention link expression of atet-activator protein to an oncogene. Since the oncogene is critical formaintaining the tumor phenotype, the linkage of tet activation tooncogene expression results in a model wherein the clones that formtumors are the same clones that are sensitive to tet activation. Thus,tumor cells will respond specifically and sensitively to doxycycline,which allows specific assessment of shRNA potency and effect. Thisapproach can be used to design/identify RNAi molecules for treatingpatients, based on identification of shRNA with most potent effectsagainst cancer cells in vivo.

Therefore, in one aspect, the invention provides an inducible nucleicacid vector comprising: a nucleic acid encoding a short hairpin RNAoperably linked to an inducible promoter, wherein the promoter isinduced in the presence of tetracycline or doxycyclin, whereinexpression of the short hairpin RNA produces siRNA which in turninhibits expression of one or more DNA replication proteins; a firstmarker gene linked to the nucleic acid encoding the short hairpin RNA,wherein the marker gene is co-expressed with the short hairpin RNA on asingle transcript, so as to allow monitoring of expression of the shorthairpin RNA; and a second marker gene that is expressed from a promoterother than the inducible promoter, so as to provide for separatemonitoring of integration of the inducible vector in a genome.

The invention provides a mouse model for use in determining the potencyof an shRNA in vivo for reducing survival of cancer cells ofchemotherapy-resistant leukemia, comprising a syngeneic mouse recipienttransplanted with tet-on competent leukemia cells, wherein said tet-oncompetent leukemia cells carry a bicistronic nucleic acid constructcomprising a promoter operably linked to a fusion gene associated withchemotherapy-resistant leukemia, and a sequence encoding a reversetet-transactivator protein, such that both coding sequences areco-expressed from said promoter.

“Tet-on competent” cells are cells comprising a bicistronic constructthat co-expresses (i) a fusion protein or other oncogene that maintainssurvival and/or growth of the leukemia cell; and (ii) a reversetet-transactivator protein.

Tet-on competent leukemia cells can be obtained from a mousetransplanted with hematopoietic stem and progenitor cells stablytransformed with the bicistronic nucleic acid construct, whereinexpression of said construct in the transplanted cells gives rise toleukemia in the mouse. In some embodiments, the hematopoietic stem andprogenitor cells are stably transformed with a nucleic acid constructcomprising a marker gene; and a sequence encoding a mutant RAS proteinbefore being transplanted into the mouse.

In some embodiments, the tet-on competent leukemia cells are stablytransformed with a second nucleic acid construct comprising: (i) atetracycline responsive first promoter operably linked to a sequenceencoding a first marker gene and a sequence encoding an shRNA and/orshRNA precursor, wherein both coding sequences are co-expressed fromsaid first promoter; and (ii) a constitutive, second promoter operablylinked to a sequence encoding a second marker gene before beingtransplanted into the syngeneic mouse recipient. Preferably, the firstand second markers provide for separate, independent monitoring ofexpression of the shRNA and/or shRNA precursor, and of integration ofsaid second nucleic acid construct.

The sequence encoding the reverse tet-transactivator protein can betranslated from an internal ribosomal entry site (IRES). Thetetracycline inducible promoter can be induced in the presence oftetracycline, doxycycline, or a tetracycline analog.

A “gene associated with chemotherapy-resistant leukemia” is a fusiongene or other oncogene characteristic of a human leukemia genotype thatconfers on leukemia cells resistance to chemotherapeutic treatment. Thefusion gene associated with chemotherapy-resistant leukemia ispreferably an MLL fusion gene, or AML1/ETO fusion gene. In particular,the fusion gene can preferably be selected from the group consisting ofMLL/ENL, MLL/AF9, AML1/ETO9a.

In a particular embodiment, the invention provides a mouse model fordetermining potency of an shRNA in vivo for inhibition of survival ofcancer cells of chemoresistant leukemia, wherein the mouse co-expressesoncogenic Nras and an MLL fusion protein and a marker protein; andwherein the mouse contains an inducible vector, comprising: a nucleicacid encoding a short hairpin RNA operably linked to an induciblepromoter, wherein the promoter is induced in the presence oftetracycline or doxycycline, wherein expression of the short hairpin RNAproduces siRNA which in turn inhibits expression of one or more DNAreplication proteins; a first marker gene linked to the nucleic acidencoding the short hairpin RNA, wherein the marker gene is co-expressedwith the short hairpin RNA on a single transcript, so as to monitorexpression of the short hairpin RNA; and a second marker gene that isexpressed from a promoter other than the inducible promoter, so as toprovide for separate monitoring of integration of the inducible vectorin a genome.

In another aspect, the invention provides an in vivo method fordetermining the potency of an shRNA for reducing survival of cancercells of chemotherapy-resistant leukemia, the method comprising:

a) providing tet-on competent leukemia cells by transplanting asyngeneic mouse with hematopoietic stem- and progenitor cells stablytransformed with a bicistronic nucleic acid construct comprising apromoter operably linked to a fusion gene associated withchemotherapy-resistant leukemia, and a sequence encoding a reversetet-transactivator protein, wherein both coding sequences areco-expressed from said promoter, and wherein expression of the constructin the transplanted cells gives rise to leukemia in the mouse, andisolating the leukemia cells from the mouse;

b) stably transforming the tet-on competent leukemia cells of step (a)with a second nucleic acid construct comprising a tetracyclineresponsive first promoter operably linked to a sequence encoding a firstmarker protein and a sequence encoding an shRNA and/or shRNA precursor,wherein both coding sequences are co-expressed from said first promoter;and a constitutive, second promoter operably linked to a sequenceencoding a second marker protein;

c) transplanting the cells of step (a) into a secondary syngeneic mouserecipient;

d) administering tetracycline, doxycycline or a tet analog to therecipient mouse of step (b) to induce expression of the shRNA and/orshRNA precursor in the leukemia cells; and

e) monitoring progression or regression of leukemia in said secondaryrecipient mouse.

The tet-on competent leukemia cells of step (a) can be stablytransformed by retroviral infection.

Progression or regression of leukemia can be monitored throughexpression of a first marker protein and/or a second marker protein. Insome instances, the bicistronic nucleic acid construct of step (a)further comprises a marker gene encoding a protein other than the firstor second marker protein encoded by the second nucleic acid construct,and progression or regression of leukemia is monitored throughexpression of said marker gene.

In some embodiments, expression of the shRNA and/or shRNA precursor inthe leukemia cells is monitored by expression of the first markerprotein encoded by the second nucleic acid construct. Integration of thesecond nucleic acid construct in the leukemia cells can be monitored byexpression of the second marker protein from said second construct.

Also provided is an in vivo method for screening a plurality of shRNAsto identify an shRNA which inhibits survival of cancer cells in vivo ina chemoresistant mouse model of acute myeloid leukemia, the methodcomprising (a) administering to the mouse one or more shRNAs and (b)determining whether the mouse of step (a) exhibits inhibition ofsurvival of cancer cells as compared to a second mouse of the mousemodel, wherein inhibition of survival of cancer cells indicates that theshRNA can be useful to treat the chemoresistant leukemia.

Also provided are in vitro and in vivo methods for screening pools ofRNAi molecules for inhibitory effects on cancer cells. The combinationof tet-on competent cancer models and tet-regulatable shRNA expressionvectors allows for monitoring shRNA expression, for example usingfluorescent and/or other reporter genes (e.g. TRMPV and derivates),which facilitates pooled shRNA negative selection screening. In suchapproaches tet-on competent cancer cells can be transduced withsequences that encode pools of shRNAs. In one embodiment, the cancercells used can be cells of the tet-on cancer models described herein(such as the MLL/AF9+Nras AML model), either in vitro or in vivo. shRNAcontaining cells can then be selected, for example by using drugselection (e.g. G418) or fluorescence-activated cell sorting (FACS).Selected cell populations harboring a library of shRNAs then can becultured in the absence or presence of doxycyline (off dox and on dox,respectively) or injected into syngeneic recipient mice that are eithertreated with doxycyline or left untreated. The representation of eachshRNA within the pool can be determined, for example by deep sequencingof shRNA cassettes in a given cell population. Based on the comparisonof shRNA representation (for example, as determined based on deepsequencing read numbers) before the assay (t0) or from cell populationsleft without doxycycline treatment (e.g. off dox), to after the assay orfrom cell populations where shRNAs were induced and the cells weresorted for shRNA expressing cells (e.g. on dox), shRNAs havinginhibitory effects can be identified. For example, shRNAs havinginhibitory effects are predicted to loose representation (show lessreads) upon shRNA induction (e.g. on dox). For example, in oneembodiment, the present invention provides an in vitro or in vivo methodfor screening pools of RNAi molecules for inhibitory effects on cancercells, the method comprising: (a) administering to a population oftet-on cancer cells a pool of sequences that encode shRNAs, (b)selecting cells that express shRNAs, (c) either culturing the cells thatexpress shRNAs in the presence or absence or presence of doxycycline, orinjecting cells that express shRNAs into syngeneic recipient mice thatare either treated with doxycyline or left untreated, (d) determiningthe representation of each shRNA within each pool before and after ofdoxycycline treatment, wherein shRNAs that have an inhibitory effect oncancer cells have lower representation after doxycycline treatment thanbefore doxycycline treatment. Variations on this scheme are apparentfrom the disclosure, including the disclosure in Example 13.

The invention also provides a method for treatingchemotherapeutic-resistant leukemia in a subject in need thereof,wherein the subject exhibits a known genotype associated with achemotherapy-resistant leukemia, the method comprising:

a) determining which known genotype associated with achemotherapy-resistant leukemia is present in a subject suffering from achemotherapy-resistant leukemia;

b) administering to the subject an RNAi molecule directed against a genewhose expression is necessary for survival of the chemotherapy-resistantleukemia cell with said known genotype, wherein the gene is selectedfrom the group consisting of: RPA3, RRM1, Rpl15, c-Myb, Bcl2, Mcl1 orMen1, so as to inhibit survival of cancer cells in the subject, andthereby treat the chemotherapeutic-resistant leukemia in the subject.

The chemotherapy-resistant leukemia can be acute myeloid leukemia.

The known genotype can comprise a chromosomal rearrangement resulting ina MLL fusion protein, or AML1/ETO fusion protein. In a preferredembodiment, the MLL or AML fusion protein is selected from the groupconsisting of MLL/ENL, MLL/AF9, AML1/ETO9a.

In some embodiments, the RNAi molecule can be directed against RPA3,Rpl15, c-Myb, Bcl2, Mcl1 or Men1. In other embodiments, the RNAimolecule can be directed against another gene expression is necessaryfor survival of the cancer cell. In one aspect, the RNAi molecule cancomprise a nucleotide sequence selected from SEQ ID NOs: 1-14 and39-190, or a portion of that sequence.

Also provided is a method for treating cancer in a subject in needthereof, the method comprising: administering to the subject an RNAimolecule directed against a gene whose expression is necessary forsurvival of the cancer cell, wherein the RNAi molecule is directedagainst a gene selected from the group of RPA3 and RRM1, so as toinhibit survival of cancer cells in the subject, and thereby treat thecancer in the subject. The RNAi molecule can comprise a nucleotidesequence selected from SEQ ID NOs: 1-14 or a portion of that nucleotidesequence. Preferably, the cancer is resistant to chemotherapeutictreatment. In preferred embodiments, the patient is diagnosed with abladder cancer or liver cancer.

In another aspect, the invention provides a method for treatingchemotherapeutic-resistant acute myeloid leukemia in a subject in needthereof, wherein the subject exhibits a protein fusion comprising MLLfused to a protein, the method comprising: determining which knowngenetic aberration characteristic of acute myeloid leukemia is presentin a subject suffering from acute myeloid leukemia; and administering tothe subject an RNAi molecule directed against a gene whose expression isnecessary for survival of the chemotherapy-resistant leukemia cell withsaid known genotype. Preferably, such gene targets and/or RNAi moleculesare validated through use of the in vivo methods of this invention fordetermining the potency of an shRNA for reducing survival ofchemotherapy-resistant leukemia cell with a known genotype. Preferably,the RNAi molecule, or the sequence encoding the shRNA comprises asequence that is selected from the group consisting of: SEQ ID NOs: 8-14and 39-190, or a portion of that nucleotide sequence, so as to inhibitsurvival of cancer cells in the subject, and thereby treat thechemotherapeutic-resistant acute myeloid leukemia in the subject.

Also provided is a method for treating a soft tissue cancer in a subjectin need thereof, wherein the subject exhibits a gene translocation ormutation associated with the soft tissue cancer, the method comprising:administering to the subject an RNAi molecule directed against a genewhose expression is necessary for survival of the soft tissue cancercell. Preferably, such gene targets and/or RNAi molecules are validatedthrough use of the in vivo methods of this invention for determining thepotency of an shRNA for reducing survival of a cancer cell with a knowngenotype. Preferably, the RNAi molecule, or the sequence encoding theshRNA comprises a sequence that is selected from the group consistingof: SEQ ID NOs: 8-14 and 39-190 or a portion of that nucleotidesequence, so as to inhibit survival of cancer cells in the subject, andthereby treat the soft tissue cancer in the subject. The soft tissuecancer can be, for example, liver cancer or lymphoma, and in someembodiments the soft tissue cancer is a chemotherapy resistant cancer.

3. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the identification of hairpins that inhibit growth ofHCT116 cultures in a proliferation assay results. HCT116 cultures in24-well plates were infected separately with amphotropic retrovirusencoding 254 different shRNAs targeting 83 cell cycle and/or E2Fregulated genes. On day 1 following infection each culture was suspendedin media containing 1.5 μg/mL puromycin to select for cells transducedwith the hairpins, then seeded to 6-well plates. On day 5 followinginfection, cells in each culture were suspended in media and countedusing a hemacytometer. Results for each culture were normalized to theaverage of the number of cells counted in cultures transduced withhairpins targeting either of two different genes not endogenous toHCT116 cells (EBNA1 and Ff-luciferase).

FIG. 2 shows hairpins that inhibited HCT116 proliferation greater thantwo-fold. The hairpin ID corresponds to Open Biosystems' catalog numberfor the respective shRNA. This can be used to access shRNA sequenceinformation from the Open Biosystems website.

FIGS. 3A-3C show growth analysis of HCT116 cultures transduced withshRNAs targeting either RPA subunits (FIG. 3A), RRM1 (FIG. 3B), PES1(FIG. 3C), or negative controls. HCT116 cultures in a 24-well plate weretransduced separately with shRNAs targeting either RPA1, RPA3, RRM1,PES1, EBNA1, or Ff-luciferase. On day 1 following transduction, eachculture was suspended in media containing 1.5 μg/mL puromycin thenseeded to 6-well plates. On day 3 following transduction, each culturewas suspended and counted using a hemacytometer. Each suspension wasdiluted and seeded to 5 wells of a E-well plate where each well wasseeded with 100,000 cells in media containing 1.5 μg/mL puromycin. In24-hour increments, one culture for each RNAi condition was suspendedand counted using a hemacytometer. HCT116 cultures transduced withshRNA's targeting either EBNA1 or Ff-luciferase were confluent by day 5after seeding.

FIGS. 4A-4C shows Western blot analysis of RPA1 (FIG. 4A), RPA2 (FIG.4B), and RPA3 (FIG. 4C) in whole cell extracts from HCT116 cellstransduced with different hairpins. On day 5 post-infection whole cellextracts (WCE's) were prepared from HCT116 cultures transduced with thenoted hairpins. Sample loading on the gel is as follows—lane 1:V2HS_(—)32105 (RPA3) WCE, lane 2: V2HS_(—)32101 (RPA3) WCE, lane 3:V2HS_(—)32160 (RPA1) WCE, lane 4: EBNA1mi1666 WCE (equivalent totalprotein loaded as the RPA3 and RPA1 RNAi WCE's), lane 5: EBNA1mi1666 WCEdiluted 1-to-2, lane 6: EBNA1mi1666 WCE diluted 1-to-4, and lane 7:EBNA1mi11666 WCE diluted 1-to-10. The antibodies used for western blotare RPA1: p70-9, RPA2: p34-20, and RPA3: Abcam ab58317. The asteriskindicates a non-specific band observed in the RPA1 western blot.

FIG. 5 shows an in vivo negative selection RNAi vector (TRMPV) thatallows for separate monitoring of retroviral integration (Venus=YFP) andshRNA expression (dsRed).

FIG. 6A shows a FACS assay at day 0 of dox treatment of lethal shRNAstudies using the TRMPV vector. in vitro.

FIG. 6B shows a FACS assay at day 3 of dox treatment of lethal shRNAstudies using the TRMPV vector. in vitro. shRNA expressing cells startto deplete for shRpa3.

FIG. 6C shows a FACS assay at day 7 of dox treatment of lethal shRNAstudies using the TRMPV vector in vitro. Almost complete depletion ofshRNA expressing cells in shRpa3 is observed. A few cells fail to induceRpa3 and grow out (Venus+, dsRed−).

FIG. 7 shows lethal shRNA studies in MLL/AF9+Nras AML.

FIGS. 8A-B shows the quantification of Hepa1.6 colony formation assaysand representative scans. (FIG. 8A) Mouse hepatocellular carcinoma cells(Hepa1.6) were transduced at high MOI with shRNAs targeting replicationgenes. Cells were plated at low density, stained with crystal violet,and staining was quantified on a Licor scanner. Values are normalized toempty vector control. (FIG. 8B) Representative scans of colony formationassays of cells infected with an antiproliferative shRNA(top—shRpa3.457) or empty vector control (bottom).

FIG. 9 shows in vitro competition assays in Hepa1.6 cells transducedwith shRNA or empty vector. Two days after transduction with aGFP-tagged vector, the number of fluorescent cells was quantified. Cellswere passaged 1:8 five times, and the number of GFPpositive transducedcells was quantified at each passage. Cells transduced with anantiproliferative shRNA (shPCNA.538 or shRpa3.457) are outcompeted byuninfected, GFP-negative cells in the population.

FIGS. 10A-F shows in vitro competition assays in MLL leukemia cells.MLL-driven leukemia cells were transduced with inducible hairpinstargeting MLL itself, MLL binding partners, and MLL target genes. Cellsexpressing the inducible shRNA (and its RFP marker) were quantified for8-11 days after doxycycline induction. In addition to positive controls(FIG. 10A—shRpa3, shMLL), only cells expressing an shRNA targeting c-Myb(FIG. 10B) display a strong proliferative disadvantage relative tountransduced cells. Knockdown of other MLL partners and targets do notaffect proliferation (FIGS. 10C-F).

FIGS. 11A-B shows identification of shRNAs that affect proliferation ofmouse liver carcinoma cells. (FIG. 11A) p53−/−RasV12 liver carcinomacells were transduced with a library of 2270 shRNAs targeting 1000genes. Relative shRNA abundance was measured by deep sequencing at 48hours after transduction and after two more weeks of passaging.Proliferative phenotype of an shRNA was assessed by its change inabundance in the cell population (log ratio of abundance at two weeks ofpassaging to abundance at 48 hours posttransduction). (FIG. 11B) Invitro competition assays were performed to validate the effectantiproliferative shRNAs (log ratio <<1). In addition to the positivecontrol (shRpa3), shRNAs targeting ribosomal components (shRpl15,shRps4x) confer a proliferative disadvantage to transduced cellsrelative to untransduced cells.

FIG. 12 shows mouse models of AML involving fusion proteins and Nras.

FIG. 13 shows a schematic of a system for negative selection RNAi inleukemia using bicistronic vectors.

FIG. 14 shows negative selection in vivo using TRMPV in bulkpopulations. Doxycycline treated mice at a terminal disease stage showno Myb-shRNA expressing cells (double positive for Venus and dsRedfluorescent markers) within the leukemia population indicating therepression of Myb represses leukemia in vivo.

FIG. 15 shows photgraphs of bone marrow histological cross-sections.Rpa3 shRNA or Myb shRNA ameliorates MLL/AF9+Nras AML.

FIG. 16 shows photgraphs of liver histological cross-sections. Knockdownby Rpa3 shRNA or Myb shRNA ameliorates MLL/AF9+Nras AML.

FIG. 17 shows quantification of tumor growth following shRpa3 inductionby doxycycline. One million mouse liver carcinoma cells transduced withan inducible shRpa3.457 were injected subcutaneously in nude mice. Twoweeks after injection, mice were either treated with doxycycline 2 mg/mlin their drinking water, or left untreated. Change in tumor volumes wasmeasured following treatment. Tumors in which shRpa3 was induced bydoxycycline stopped growing or regressed.

FIG. 18 shows luciferase imaging of leukemic mice harboring inducibleshRpa3. Sublethally irradiated recipient mice were transplanted with1×10⁶ cells from a luciferase-tagged, chemoresistant leukemia harboringinducible shRpa3. Mice were either left untreated, or treated withdoxycycline at an early (day 18 postinjection) or late (day 21) stage ofdisease progression. While untreated mice succumbed to the disease,shRpa3 induction by doxycycline caused full remission in both early andlate treatment groups.

FIGS. 19A-B shows luciferase imaging of leukemic mice with inducibleshMyb. Sublethally irradiated recipient mice were transplanted with1×10⁶ cells from a luciferase-tagged, chemoresistant MLLdriven leukemiaharboring an inducible shRNA targeting c-Myb. Mice were either leftuntreated, or treated with doxycycline. While untreated mice succumbedto the disease, shMyb induction by doxycycline resulted in full,longterm remission. (FIG. 19A). Kaplan meyer survival curves of micetransplanted with clonal MLL/AF9+Nras induced leukemia harboring shRNAstargeting Myb or Braf. Induction of shRNAs by doxycycline treatmentinduces long-term survival advantage or cure in mice harboring MybshRNAs, but has no effect in mice harboring Braf shRNAs. Mice remaindisease-free after discontinuing doxycycline treatment after 32 days.(FIG. 19B)

FIG. 20 shows a schematic of a strategy for using negative selectionRNAi screening to identify epigenetic modifiers as drug targets forchemotherapy resistant AML using a custom epigenetic library of 1,100shRNAs targeting genes for 235 known epigenetic regulators. Forscreening, these shRNAs were cloned into the LMN mir30-embedded shRNAvector.

FIG. 21 shows results of a primary screen of each of the 1100 individualshRNAs in the epigenetic library in chemotherapy resistant AML. EachshRNA was monitored for its ability to confer a proliferativedisadvantage to MLL-AF9/Nras leukemia cells in vitro. The primary screenidentified 35 epigenetic regulators (129 shRNAs) that were required forproliferation of leukemia cells in vitro.

FIG. 22 shows a secondary screen of 129 shRNAs directed against 35epigentic regulators to determine the ability of the identified shRNAsto selectively inhibit growth of leukemia cells. The screening revealed8 genes, which leukemias, in contrast to three non-transformed cells,selectively require for their proliferation.

FIGS. 23A-D shows results of a secondary screen identifying 8 geneswhich leukemias selectively require for their proliferation. The abilityof shRNAs directed against these 8 genes, and of shRNAs directed againstRpa3 and Myc genes, to inhibit growth was compared in leukemia cells(FIG. 23A), non-transformed erythroid cells (G1E) (FIG. 23C),non-transformed myeloid cells (32D) (FIG. 23B) and non-transformedstem-like cells (EML) (FIG. 23D).

FIG. 24 shows the system and strategy using tet-on competent AML cellsand the TRMPV vector to perform negative selection RNAi screening invivo to validate the therapeutic potential of inhibiting epigenticmodifier genes identified by in vitro screening as required forproliferation of leukemia cells. Each shRNA targeting an epigeneticmodifier gene is cloned into the TRMPV vector and introduced into tet-onleukemia cells. Following selection, the leukemia cells weretransplanted into recipient mice, which are then treated withDoxycycline 3 days after transplant. The TRMPV/luciferase tagged tet-onAML system facilitates monitoring of shRNA expressing cells in vivo viabioluminescence, providing a rapid and robust assay to assesstumor-inhibitory RNAi effects.

FIG. 25 shows Kaplan-Meyer survival curves of lethally irradiatedrecipient mice, which were reconstituted with MLL-AF9/Nras tet-on AMLcells transduced with tet-inducible shRNAs targeting epigenetic modifiergenes (EED, Aof2, Suz12, Men1 and SMARCD1) and Rpa3. Inhibition of thesegene targets by these shRNA, as compared to a Renilla luciferase controlshRNA, revealed a survival benefit or substantial rate of cure in vivo.

FIG. 26 shows a FACS analysis of shRNA expression in bone marrow atterminal disease stage, demonstrating that shRNAs targeting epigeneticmodifier genes (EED, Aof2, Suz12, Men1 and SMARCD1) inhibitedproliferation of leukemia cells as the disease developed, compared to aRenilla luciferase control.

FIG. 27 shows a schematic of a strategy using a dual-color competitionassay enabling a direct and robust assessment of shRNA effects in thereconstitution of hematopoietic tissues in vivo. LMN vectors harboringshRNAs are transduced into fetal livers. Infected cells are subsequentlytransplanted into lethally irradiated recipients. This assay comparesthe relative contribution of the experimental shRNA (in an LMN vectorharboring a GFP marker) to a control, neutral shRNA (in an LMN vectorharboring a red fluorescent protein marker (mCherry)). The effect of theexperimental shRNA on hematopoiesis is assessed by the ratio of thesetwo markers in subsets of peripheral blood cells, or bone marrow orspleen, measured at 4 weeks.

FIG. 28 shows the results of a dual-color competition assay performed toassess the effect of various experimental shRNAs on hematopoiesis invivo, as compared to a luciferase control shRNA.

FIG. 29 shows a comparison of the inhibitory effect of differentconcentrations of tranylcypromine (Aof2 inhibitor) on proliferation ofMLL-AF9/Nras leukemia cells other human AML cell lines and anon-transformed myeloid cells (32D) in vitro. The results indicated thattranylcypromine has a selective inhibitory effect on proliferation ofAML cells

FIGS. 30A-H shows a strategy for generation of genetically definedmosaic mouse models based on common genetic associations in human AML.Frequency of Nras mutations in 111 cases of pediatric AML depending onmajor karyotype groups. Nras mutations are especially common in concertwith core-factor binding translocations (AML1/ETO, CBFbeta/MYH11) andMLL rearrangements. (FIG. 30A). Kaplan-Meier plot showing the overallsurvival of pediatric AML patients treated after 1998 depending oncertain karyotype abnormalities including t(8;21)=AML1/ETO (n=10),inv(16)/t(16;16)=CBF/MYH11 (n=9), 11q23/MLL rearrangements (n=9)compared to other subtypes (n=22, excluding patients with PML/RARpositive AML). The presence of AML1/ETO and MLL fusion proteins hasopposite effects on long-term therapy outcome (FIG. 30B). MSCV-basedretroviral constructs used to co-express AML oncogenes with fluorescentand bioluminescent markers (FIG. 30C). Schematic overview of mosaic AMLmouse models. Wildtype C57BL/6 fetal liver cells (FLC) isolated at ED13.5-15.5 were (co-)transduced with oncogenic retroviruses and used toreconstitute the hematopoietic system of lethally irradiated recipientmice (FIG. 30D). Mice reconstituted with FLCs transduced with theindicated transgenes were monitored for illness and died or wereeuthanized at a terminal disease stage. The data are presented in aKaplan-Meier format showing the percentage of mouse survival at varioustimes post transplantation (FIG. 30E). Luciferase imaging of recipientmice of FLCs transduced with the indicated genes at 14, 21 and 42 daysfollowing transplantation. Transduction of Luciferase-IRES-Nras rapidlyinduces onset of Luciferase-positive disease only in concert withAML1/ETO9a or MLL/ENL (FIG. 30F). Expression analysis of retroviraloncogenes in wildtype bone marrow (wt bm), and independent primary AMLs(1-3) with indicated genotypes. Expression of human AML1/ETO9a andMLL/ENL transcripts was verified by RT-PCR using fusion-site specificprimers. No reverse transcriptase controls were negative in all samples(not shown). Western-blot analysis using pan-Ras and Nras-specificantibodies demonstrating Nras overexpression on leukemia lysates derivedfrom Nras-cotransduced FLCs. Overall Ras levels (pan-Ras) do not showsignificant elevation (FIG. 30G). Baseline phospho-Erk levels arestrongly elevated in leukemias deriving from Nras-cotransduced FLCs.Levels of phosphorylated Erk were measured using phospho-specific flowcytometry in wildtype whole bone marrow and GFP-positive MLL/ENL,MLL/ENL+Nras and AML1/ETO9a+Nras leukemias. Representative histogramsare shown (FIG. 30H).

FIGS. 31A-D shows results indicating that defined mosaic AML mousemodels have genotype-dependent morphology consistent with human AML.May-Grünwald-Giemsa stained peripheral bloodsmears (20 foldmagnification) (FIG. 31A). Wright-Giemsa stained bone marrowcytocentrifugation (100 fold magnification) predominantly show immatureblasts in AML1/ETO9a+Nras leukemia, while MLL/ENL+Nras leukemia ischaracterized by more mature myelomonocytic cells at variousdifferentiation levels (FIG. 31B). Bone marrow immunphenotyping inAML1/ETO9a+Nras leukemic mice shows infiltration ofGFP+/c-Kit+/Mac-1−/Gr-1− immature blasts, while MLL/ENL+Nras bone-marrowis dominated by GFP+/c-Kit−/Mac-1+/Gr-1+ myelomonocytic cells (FIG.31C). Hematoxylin-eosin stained liver sections showing massive leukemicinfiltration (20 fold magnification, scale bar 100 um) (FIG. 31D).

FIGS. 32A-C shows that AML1/ETO9a+Nras and MLL/ENL+Nras AMLs showdramatic differences in their response to combined chemotherapy in vivo.Luciferase-imaging and histological analysis of hematoxylin-eosinstained bone marrow sections before (d0), during (d3) and after 5 daysof chemotherapy (d6) show therapy-triggered regression and ultimatelycomplete remission of AML1/ETO9a+Nras leukemia (left panel), whileMLL/ENL+Nras leukemia only show decelerated progression, withpersistance of blasts in response to treatment (right panel). 40 foldmagnification; scale bars 50 um (FIG. 32A). Long-term follow-upluciferase imaging of untreated and treated AML1/ETO9a+Nras leukemia.Treated mice achieve durable remissions lasting at least 30 days (d30).While most mice subsequently relapse, some mice remain in remissionfollowing chemotherapy (d60) (FIG. 32B). Kaplan-Meier survival curves ofuntreated and treated AML1/ETO9a+Nras and MLL/ENL+Nras mice followingthe initiation of chemotherapy (FIG. 32C).

FIGS. 33A-E shows in vivo expression analysis of immediate chemotherapyresponse programs, which identifies differences in p53 induction betweenAML1/ETO9a+Nras and MLL/ENL+Nras AMLs. AML1/ETO9a+Nras leukemias showcomplex gene expression changes in response chemotherapy, while thisresponse profile is blunted in the MLL/ENL+Nras context. For twoindependent primary leukemias (1,2) of each genotype the expressionprofile 2 h after combined chemotherapy (a single dose of Ara-C andDoxorubicin i.p.) was compared to this of an untreated control. For eachcondition microarray profiles were acquired in two technical replicates(a,b). Treatment-induced expression changes were rendered in agreen-black-red pseudo color scheme for all genes with an average foldchange ≧2.0 in either genotype (FIG. 33A). KEGG pathways analysis of theAML1/ETO9a+Nras specific drug-response signature identifies fivepathways with significant alteration and reveals differences in p53response levels. The 398 most significantly altered genes in treatedAML1/ETO9a+Nras leukemia were identified by SAM (FDR 0.2, FC>2) andinferred with KEGG pathways (Ogata et al. 1999) using DAVID (Dennis etal. 2003) (FIG. 33B). Quantitative real-time PCR analysis of p21 andMdm2 at various time points after chemotherapy in vivo. Baselineexpression and induction of both p53 target gene transcripts isattenuated in MLL/ENL+Nras leukemia (FIG. 33C). Western-blot analysis ofp53 and p21 in leukemic spleens (>85% GFP+ infiltration) at various timepoints after i.p. administration of one dose of combined chemotherapy.AML1/ETO9a+Nras AML show much stronger p53 induction resulting in astronger and more durable induction of its target p21 (FIG. 33D).Western-blot analysis of p53 and p21 in recipient mice transplanted withindependent primary AMLs were either left untreated (−) or were treatedwith one dose of combined chemotherapy 4 hours prior to sample harvest(+). Individual differences in p53 drug-response programs are dependenton the AML genotype (FIG. 33E).

FIGS. 34A-C shows results of studies in the AML mouse model, indicatingthat loss of p53 dramatically accelerates AML1/ETO9a, but does notaffect MLL/ENL induced leukemogenesis. Kaplan-Meier survival curves oflethally irradiated recipient mice, which were reconstituted withwildtype or p53−/− FLCs transduced with either AML1/ETO9a or MLL/ENL.Loss of p53 accelerates AML1/ETO9a, but not MLL/ENL inducedleukemogenesis (FIG. 34A). Bone marrow immunophenotyping of wildtype andp53-deficient AML1/ETO9a and MLL/ENL induced leukemias. Loss of p53 doesnot affect the typical disease morphology induced by both fusionproteins (FIG. 34B). Kaplan-Meier survival curves of lethally irradiatedrecipient mice, which were reconstituted with wildtype or p53−/− FLCsco-transduced with AML1/ETO9a and NrasG12D. Loss of p53 also acceleratesAML1/ETO9a+Nras induced leukemogenesis (FIG. 34C).

FIGS. 35A-D shows results of studies in the AML mouse model onmechanisms of chemoresistance in AML indicating that loss of p53 induceschemotherapy resistance in AML1/ETO9a+Nras AML. Luciferase imaging ofAML1/ETO9a+Nras leukemias generated in wildtype (left panel) or p53−/−(right panel) FLCs before (d0), during (d3) and after chemotherapy (d6,d24). Recipient mice of p53-deficient AML1/ETO9a+Nras leukemias retainbioluminescent signal under therapy (FIG. 35A). Hematoxylin-eosinstained bone marrow sections of recipients of p53-deficientAML1/ETO9a+Nras leukemia at various time points following chemotherapydemonstrating blast persistence under chemotherapy (40 foldmagnification; scale bars 50 um) (FIG. 35B). GFP histograms of bonemarrow flow cytometry before (d0) and at various time points during (d3)and after (d6, d9) chemotherapy. While AML1/ETO9a+Nras blasts harboringwildtype p53 (AE+Nras p53 wt) rapidly clear, both MLL/ENL+Nras (ME+Nrasp53 wt) and p53-deficient AML1/ETO9a+Nras leukemias (AE+Nras p53−/−)show persistence of GFP+ cells in bone marrow (FIG. 35C). Kaplan-Meiersurvival curves of untreated and treated recipient mice of p53 wildtypeand p53-deficient AML1/ETO9a+Nras leukemias. Loss of p53 impedes thelong-term outcome of chemotherapy (FIG. 35D).

FIGS. 36A-D shows an outline of the strategy used to establish acombined chemotherapy regimen in the mouse AML model modeling clinicalAML induction therapy in human patients. Schematic overview of thechemotherapy protocol. The regimen involves daily i.p. injections ofCytarabine (100 mg/kg/d over 5 days) and Doxorubicin (3 mg/kg/d over 3days) (FIG. 36A). Bioluminescent imaging and peripheral blood smears(May-Grünwald-Giemsa stained) of recipient mice ofMLL/ENL-IRES-Luciferase+FLT3-ITD induced AML with and withoutchemotherapy. Treatment is initiated upon detection of clear signals inpelvis, tail and both femurs and early hepatosplenic infiltration, whichwas ˜5d before leukemia became detectable in peripheral blood smears.Treatment induces peripheral leucopenia, a deceleration of diseaseprogression, while luciferase imaging demonstrates blast persistence inbone marrow of MLL/ENL+FLT3 AML recipient mice as compared to controls(FIGS. 36B-C). Kaplan-Meier survival curves of untreated and treatedrecipient mice of MLL/ENL-IRES-Luciferase+FLT3-ITD induced AML (FIG.36D).

FIG. 37 shows a schematic of pooled negative selection screening intet-on competent MLL/AF9+Nras AML using a TRMPV shRNA pool comprising1230 shRNAs (64 controls, 1166 experimental shRNAs). The shRNArepresentation is analyzed by deep sequencing in the initial plasmidpool (P0), after transduction and drug selection (T0), with or withouttreatment with doxycycline after passaging in cell culture (ON/OFF) ortransplantation and expansion in vivo (VON/VOFF).

FIG. 38 shows a further schematic of the pooled negative selectionscreening method. In this method, samples on dox are sorted for shRNAexpressing cells (Venus/dsRed double-positive) prior to DNA isolationand PCR to purify for cells that are exposed to specific shRNA effects.This step is critical to the success of screening and is facilitated bythe use of TRMPV and tet-on competent cancer models.

FIG. 39 shows a schematic of parallel pooled negative selectionscreening in different tet-on competent cancer models and normalreference cells. Such screens are focused on identifying shRNAs thatspecifically inhibit cancer cells, either generally or in certaingenetic contexts.

FIG. 40 shows 64 shRNAs that were previously analyzed for inhibitoryeffects in MLL/AF9+Nras AML and Mefs and are used in the pooled negativeselection screening method as controls.

FIG. 41 shows results of Solexa-deep sequencing of the TRMPV plasmidpool before addition of the 64 control shRNAs.

FIG. 42 shows a correlation plot of Solexa deep sequencing readscomparing the representation of individual shRNAs in the plasmid pool(P) and the transduced and selected cell population (T0). R denotes thePearson product-moment correlation coefficient.

FIG. 43 shows a correlation plot of Solexa deep sequencing readscomparing the representation of individual shRNAs before (T0) and afternine passages in cell culture (OFF). R denotes the Pearsonproduct-moment correlation coefficient.

FIG. 44 shows a correlation plot of Solexa deep sequencing readscomparing the representation of individual shRNAs in two independentreplicates after nine passages in cell culture (OFF1, OFF2). R denotesthe Pearson product-moment correlation coefficient.

FIG. 45 shows a correlation plot of Solexa deep sequencing readscomparing the representation of individual shRNAs before (T0) and aftertransplantation into syngeneic recipient mice and leukemia development(VOFF). R denotes the Pearson product-moment correlation coefficient. Rdenotes the Pearson product-moment correlation coefficient.

FIG. 46 shows a correlation plot of Solexa deep sequencing readscomparing the representation of individual shRNAs in two independentreplicates after transplantation into syngeneic recipient mice andleukemia development (VOFF1, VOFF2). R denotes the Pearsonproduct-moment correlation coefficient.

FIG. 47 shows a correlation plot of Solexa deep sequencing readscomparing the representation of individual shRNAs in two independentreplicates sorted for shRNA expressing cells after nine passages in cellculture under doxycycline treatment (ON1, ON2). R denotes the Pearsonproduct-moment correlation coefficient.

FIG. 48 shows a correlation plot of Solexa deep sequencing readscomparing the representation of individual shRNAs in two independentreplicates after transplantation into syngeneic recipient mice,doxycycline treatment and leukemia development (VON1, VON2). Sampleswere harvested from leukemic mice and sorted for cells with sufficientshRNA expression. R denotes the Pearson product-moment correlationcoefficient.

FIG. 49 shows the relative representation (read numbers in Solexa deepsequencing) in doxycycline treated MLL/AF9+Nras leukemia samples (OnDox) compared to the initial representation (T0). Negative controlshRNAs are indicated as dark bars. None of the negative control shRNAsfalls into the scoring window of >8 fold depletion.

FIG. 50 shows the relative representation of generally lethal controlshRNAs in doxycycline treated MLL/AF9+Nras leukemia samples (On Dox)compared to the initial representation (T0). 10 out of 11 lethal shRNAsare found depleted more than 8 fold.

FIG. 51 shows the relative representation of shRNAs known tospecifically deplete MLL/AF9+Nras leukemia in doxycycline treatedleukemia samples (On Dox) compared to the initial representation (T0). 8out of 11 of these shRNAs are found depleted more than 8 fold.

4. DETAILED DESCRIPTION OF THE INVENTION

RNAi technology enables specific suppression of the expression ofvirtually any gene. However, to obtain functional RNAi reagents forclinical applications, it is advantageous to identify a strategy forrational drug design that allows both gene targets and RNAi molecules tobe functionally validated in a defined genetic context in vivo thatreflects human disease. Here we provide a mouse model that can be usedto design rational cancer therapies based on the particular genotype ofcancer cells found in human cancers, and in particular, those found inhuman AML patients. Empirically, this model has demonstrated remarkablesimilarity with human AML patients in the genotype-response pattern tostandard induction chemotherapy, implying that such a system can predictthe behavior of therapeutic agents in the clinic.

We additionally provide defined, tet-on competent, mouse leukemia modelsexpressing inducible shRNAs to identify particular RNAi moleculesexhibiting the most potent in vivo efficacy for therapeutically curingchemoresistant leukemia. In particular, we apply tet-regulated in vivoRNAi to identify candidate drug target genes and RNAi molecules directedagainst such drug target genes for therapeutic treatment ofchemoresistant AML and other leukemias. We additionally use thesesystems to further extend observations of in vitro RNAi screens andprovide in vivo validation for drug target genes and RNAi moleculesidentified through such in vitro screens, in particular target genesencoding certain DNA replication proteins.

4.1 GENERAL DEFINITIONS

A “coding sequence” or a sequence “encoding” a particular molecule is anucleic acid that is transcribed (in the case of DNA) or translated (inthe case of mRNA) into a polypeptide or inhibitory RNA (e.g., an shRNAor an antisense), in vitro or in vitro, when operably linked to anappropriate regulatory sequence. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxy) terminus. A coding sequencecan include, but is not limited to, cDNA from prokaryotic or eukaryoticmRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, andsynthetic DNA sequences. A transcription termination sequence willusually be located 3′ to the coding sequence.

The term “gene” refers to a nucleic acid comprising an open readingframe encoding a polypeptide, including both exon and (optionally)intron sequences. The nucleic acid can also optionally includenon-coding sequences such as promoter and/or enhancer sequences.

“Nucleic acid” refers to polynucleotides such as deoxyribonucleic acid(DNA) and ribonucleic acid (RNA). The term can include single-strandedand double-stranded polynucleotides.

“Operably linked” means that the coding sequence is linked to aregulatory sequence in a manner which allows expression of the codingsequence. Regulatory sequences include promoters, enhancers, and otherexpression control elements that are art-recognized and are selected todirect expression of the coding sequence.

A “small molecule” is a compound having a molecular weight less thanabout 2500 amu, in particular less than about 2000 amu, even moreparticular less than about 1500 amu. In one embodiment, the molecularweight less is less than about 1000 amu, or less than about 750 amu.

A “subject” or “patient” can be a human or non-human animal.

A “transduced cell” is one that has been genetically modified. Geneticmodification can be stable or transient. Methods of transduction (i.e.,introducing vectors or constructs into cells) include, but are notlimited to, liposome fusion (transposomes), viral infection, and routinenucleic acid transfection methods such as electroporation, calciumphosphate precipitation and microinjection. Successful transduction willhave an intended effect in the transduced cell, such as gene expression,gene silencing, enhancing a gene target, or triggering targetphysiological event.

In one embodiment, “treating” means slowing, stopping or reversing theprogression of a disease or disorder. “Treating” can also meanamelioration of symptoms associated with a disease or disorder.

“Vector” refers to a vehicle for introducing a nucleic acid into a cell.Vectors include, but are not limited to, plasmids, phagemids, viruses,bacteria, and vehicles derived from viral or bacterial sources. A“plasmid” is a circular, double-stranded DNA molecule. A useful type ofvector for use in the present invention is a viral vector, whereinheterologous DNA sequences are inserted into a viral genome that can bemodified to delete one or more viral genes or parts thereof. Certainvectors are capable of autonomous replication in a host cell (e.g.,vectors having an origin of replication that functions in the hostcell). Other vectors can be stably integrated into the genome of a hostcell, and are thereby replicated along with the host genome.

4.2 RNAI MECHANISM

Investigation of the role of DNA replication genes in modifying humancancer cell proliferation can be facilitated by specifically silencing aparticular gene of interest. One such silencing means is through RNAinterference (RNAi). RNAi stems from a phenomenon observed in plants andworms, whereby double-stranded RNA (dsRNA) blocks gene expression in aspecific and post-transcriptional manner. The dsRNA is cleaved by anRNAse III enzyme “DICER” into a 21-23 nucleotide small interfering RNA(siRNA). These siRNAs are incorporated into an RNA-induced silencingcomplex (RISC) that identifies and silences RNA complementary to thesiRNA. Without being bound by theory, RNAi appears to involve silencingof cytoplasmic mRNA by triggering an endonuclease cleavage, by promotingtranslation repression, or possibly by accelerating mRNA decapping(Valencia-Sanchez et al., 2006, Genes & Devel. 20: 515-524). Biochemicalmechanisms of RNAi are currently an active area of research.

Three mechanisms of utilizing RNAi in mammalian cells have beendescribed. The first is cytoplasmic delivery of siRNA molecules, whichare either chemically synthesized or generated by DICER-digestion ofdsRNA. These siRNAs are introduced into cells using standardtransfection methods. The siRNAs enter the RISC to silence target mRNAexpression.

The second mechanism is nuclear delivery, via viral vectors, of geneexpression cassettes expressing a short hairpin RNA (shRNA). The shRNAis modeled on micro interfering RNA (miRNA), an endogenous trigger ofthe RNAi pathway (Lu et al., 2005, Advances in Genetics 54: 117-142,Fewell et al., 2006, Drug Discovery Today 11: 975-982). The endogenousRNAi pathway is comprised of three RNA intermediates: a long, largelysingle-stranded primary miRNA transcript (pri-mRNA); a precursor miRNAtranscript having a stem-and-loop structure and derived from thepri-mRNA (pre-miRNA); and a mature miRNA. The miRNA is transcribed by anRNA polymerase II promoter into the pri-mRNA transcript, which is thencleaved to form the pre-miRNA transcript (Fewell et al., 2006). Thepre-miRNA is transported to the cytoplasm and is cleaved by DICER toform mature miRNA. miRNA then interacts with the RISC in the same manneras siRNA. shRNAs, which mimic pre-miRNA, are transcribed by RNAPolymerase II or III as single-stranded molecules that form stem-loopstructures. Once produced, they exit the nucleus, are cleaved by DICER,and enter the RISC as siRNAs.

The third mechanism is identical to the second mechanism, except thatthe shRNA is modeled on primary miRNA (shRNAmir), rather than pre-miRNAtranscripts (Fewell et al., 2006). An example is the miR-30 miRNAconstruct. The use of this transcript produces a more physiologicalshRNA that reduces toxic effects. The shRNAmir is first cleaved toproduce shRNA, and then cleaved again by DICER to produce siRNA. ThesiRNA is then incorporated into the RISC for target mRNA degradation.

To date, distinct forms of RNA silencing have been found to regulategene expression, to mediate antiviral responses, to organize chromosomaldomains, and to restrain the spread of selfish genetic elements. Forexample, miRNAs derived from dsRNA precursors regulate gene expressionin somatic cells by reducing translation and stability of protein-codingmRNAs.

The primary step in miRNA biogenesis is the nuclear cleavage of the“primary micro RNA” (pri-miRNA), liberating an approximately 70nucleotide (nt) stem-loop intermediate known as “micro RNA precursor”(pre-miRNA). This processing step is performed by the RNase IIIendonuclease Drosha, in conjunction with the dsRNA-binding protein“DiGeorge syndrome Critical Region gene 8” (DGCR8) in humans (Pasha inDrosophila), leading to 5′ monophosphates and ˜2 nt 3′ overhangs,characteristic for RNase III endonucleases.

The pre-miRNAs are then actively transported to the cytoplasm byExportin-5 and the Ran-GTP cofactor. Subsequently, the mature miRNAs areexcised by another RNase III endonuclease Dicer, acting together withthe dsRNA-binding protein tar-binding protein (TRBP) in humans orLoquacious (Logs) in flies. Depending on the species, the resultingshort dsRNAs are about 21 to 28 nts in length.

For mRNA degradation, translational repression, or deadenylation, maturemiRNAs or siRNAs are loaded into the RNA Induced Silencing Complex(RISC) by the RISC-loading complex (RLC). Subsequently, the guide strandleads the RISC to cognate target mRNAs in a sequence-specific manner andthe Slicer component of RISC hydrolyses the phosphodiester boundcoupling the target mRNA nucleotides paired to nucleotide 10 and 11 ofthe RNA guide strand. Slicer forms together with distinct classes ofsmall RNAs the RNAi effector complex, which is the core of RISC.Therefore, the “guide strand” is that portion of the double-stranded RNAthat associates with RISC, as opposed to the “passenger strand,” whichis not associated with RISC. The target sequence contained in a reporterconstruct of the present invention is at least partially complementaryto at least a portion of the guide strand.

To further accelerate the study of cancer genes in vivo, stable RNAitechnology has been used to successfully identify and suppress targetgenes associated with tumorigenesis. For example, expression ofmicroRNA-based shRNA specific to Trp53 produces “potent, stable, andregulatable gene knock-down in cultured cells . . . even when present ata single copy in the genome” (Dickins et al., 2005, Nat. Genet. 37:1289-1295). Tumors induced by the p53 knockdown regress uponre-expression of Trp53. (Dickins et al., 2005). The suppression of theTrp53 gene expression by shRNA is also possible in stem cells andreconstituted organs derived from those cells (Hemann et al., 2003, Nat.Genet. 33: 396-400). Moreover, the extent of inhibition of p53 functionby the shRNA correlates with the type and severity of subsequentlymphomagenesis (Hemann et al., 2003).

RNAi is a powerful tool for in vitro and in vivo studies of genefunction and for therapy in both human and veterinary contexts.Depending on the application, any type of RNAi, including but notlimited to si- or shRNAs, can be used as RNAi triggers. siRNAs have theadvantage of being directly transfectable, chemically synthesizedoligonucleotides that circumvent the need for cloning. siRNAs enter themiRNA processing pathway at a later stage, and bypass Drosha processing,Exportin-5 export, and, depending on their size, Dicer cleavage.However, when the objective is therapeutic, it can be useful to usemiRNA-based shRNAs as they tend to yield more effective silencing (Changet al., Nature Methods, 2006, 3: 707-714). In addition, the small sizeof si- and shRNAs, compared with traditional antisense molecules,prevents activation of the dsRNA-inducible interferon system present inmammalian cells. This helps avoid the non-specific phenotypes normallyproduced by dsRNA larger than 30 base pairs in somatic cells. See, e.g.,Elbashir et al., 2002, Methods Enzymol. 26: 199-213; McManus and Sharp,2002, Nature Reviews 3: 737-747; Hannon, 2002, Nature 418: 244-251;Brummelkamp et al., 2002, Science 296: 550-553; Tuschl, 2002, NatureBiotechnology 20: 446-448; U.S. Publication No. 2002/0086356; WO99/32619; WO 01/36646; and WO 01/68836.

As discussed above, RNAi can be achieved using microRNA-based shRNAsthat can be potent triggers of the RNAi machinery and are capable ofefficiently suppressing gene expression when expressed from a singlecopy in the genome (Dickins et al., 2005; Silva et al., 2005, Nat.Genet. 37: 1281-1288). This technology has been used in the mosaic mousemodel of hepatocellular carcinoma (HCC) to show that stable knockdown ofthe Trp53 tumor suppressor by RNAi can mimic Trp53 gene loss in vivo(Zender et al., 2005), and that regulated RNAi can reversibly modulateendogenous p53 expression to implicate the role of p53 loss in tumormaintenance (Xue et al., 2007, Nature 445: 656-660). Similar approacheshave been used to rapidly validate Deleted in Liver Cancer 1 (DLC1) as apotent tumor suppressor gene (Xue et al., 2008, Genes Devel. 22:1439-1444).

RNAi is possible via direct introduction of siRNA into the cell, or bygene expression cassettes expressing shRNA or shRNAmir. shRNA andshRNAmir are modeled on intermediate constructs of miRNA. Both arecleaved by DICER to form siRNAs and interact with the RISC complex inthe same manner as siRNA.

4.3 RNAI MOLECULES

Interfering RNA or small inhibitory RNA (RNAi) molecules include shortinterfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), andmicro-RNAs (miRNAs) in all stages of processing, including shRNAs,pri-miRNAs, and pre-miRNAs. These molecules have different origins:siRNAs are processed from double-stranded precursors (dsRNAs) with twodistinct strands of base-paired RNA; siRNAs that are derived fromrepetitive sequences in the genome are called rasiRNAs; miRNAs arederived from a single transcript that forms base-paired hairpins. Basepairing of siRNAs and miRNAs can be perfect (i.e., completelycomplementary) or imperfect, including bulges in the duplex region.

RNAi molecules useful in this invention can be, without limitation,shRNA, siRNA, piwi-interacting RNA (piRNA), micro RNA (miRNA),double-stranded RNA (dsRNA), antisense RNA, or any other RNA speciesthat can be cleaved inside a cell to form interfering RNAs. As usedherein, siRNAs useful in this invention include, without limitation,modified siRNAs, including siRNAs with enhanced stability in vivo.Modified siRNAs include molecules containing nucleotide analogues,including those molecules having additions, deletions, and/orsubstitutions in the nucleobase, sugar, or backbone; and molecules thatare cross-linked or otherwise chemically modified. (See Crooke, U.S.Pat. Nos. 6,107,094 and 5,898,031; Elmen et al., U.S. Publication Nos.2008/0249039 and 2007/0191294; Manoharan et al., U.S. Publication No.2008/0213891; MacLachlan et al., U.S. Publication No. 2007/0135372; andRana, U.S. Publication No. 2005/0020521; all of which are herebyincorporated by reference.)

As used herein, an “shRNA molecule” includes a conventional stem-loopshRNA, which forms a precursor miRNA (pre-miRNA). “shRNA” also includesmicro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strandand the passenger strand of the miRNA duplex are incorporated into anexisting (or natural) miRNA or into a modified or synthetic (designed)miRNA. When transcribed, an shRNA forms a primary miRNA (pri-miRNA) or astructure very similar to a natural pri-miRNA. The pri-miRNA issubsequently processed by Drosha and its cofactors into pre-miRNA.Therefore, the term “shRNA” includes pri-miRNA (shRNA-mir) molecules andpre-miRNA molecules.

A “stem-loop structure” refers to a nucleic acid having a secondarystructure that includes a region of nucleotides which are known orpredicted to form a double strand or duplex (stem portion) that islinked on one side by a region of predominantly single-strandednucleotides (loop portion). The terms “hairpin” and “fold-back”structures are also used herein to refer to stem-loop structures. Suchstructures are well known in the art and the term is used consistentlywith its known meaning in the art. As is known in the art, the secondarystructure does not require exact base-pairing. Thus, the stem caninclude one or more base mismatches or bulges. Alternatively, thebase-pairing can be exact, i.e. not include any mismatches.

In some instances the precursor miRNA molecule can include more than onestem-loop structure. The multiple stem-loop structures can be linked toone another through a linker, such as, for example, a nucleic acidlinker, a miRNA flanking sequence, other molecule, or some combinationthereof.

MicroRNAs are endogenously encoded RNA molecules that are about22-nucleotides long and generally expressed in a highly tissue- ordevelopmental-stage-specific fashion and that post-transcriptionallyregulate target genes. More than 200 distinct miRNAs have beenidentified in plants and animals. These small regulatory RNAs arebelieved to serve important biological functions by two prevailing modesof action: (1) by repressing the translation of target mRNAs, and (2)through RNA interference (RNAi), that is, cleavage and degradation ofmRNAs. In the latter case, miRNAs function analogously to smallinterfering RNAs (siRNAs). The highly tissue-specific or developmentallyregulated expression of miRNAs is likely key to their predicted roles ineukaryotic development and differentiation. Analysis of the endogenousrole of miRNAs is facilitated by techniques that allow the regulatedover-expression or inappropriate expression of authentic miRNAs in vivo.The ability to regulate the expression of siRNAs will greatly increasetheir utility both in cultured cells and in vivo. Thus, one can designand express artificial miRNAs based on the features of existing miRNAgenes.

Short hairpin RNAs can be designed to mimic endogenous miRNAs. ManymiRNA intermediates can be used as models for shRNA or shRNAmir,including without limitation a miRNA comprising a backbone design ofmiR-15a, -16, -19b, -20, -23a, -27b, -29a, -30b, -30c, -104, -132s,-181, -191, -223 (see U.S. Publication No. 2005/0075492). The miR-30natural configuration has proven especially beneficial in producingmature synthetic miRNAs. miR30-based shRNAs and shRNAmirs have complexfolds, and, compared with simpler stem/loop style shRNAs, are morepotent at inhibiting gene expression in transient assays. Moreover, theyare associated with less toxic effects in cells.

In a certain embodiment, shRNA molecules are designed based on the humanmiR-30 sequence, redesigned to allow expression of artificial shRNAs bysubstituting the stem sequences of the pri-miR-30 with unrelatedbase-paired sequences (Siolas et al., 2005, Nat. Biotech. 23: 227-231;Silva et al., 2005, Nat. Genet. 37: 1281-1288); Zeng et al. (2002),Molec. Cell 9: 1327-1333). The natural stem sequence of the miR-30 canbe replaced with a stem sequence from about 16 to about 29 nucleotidesin length, in particular from about 19 to 29 nucleotides in length. Theloop sequence can be altered such that the length is from about 3 toabout 23 nucleotides. In one embodiment, the stem of the shRNA moleculeis about 22 nucleotides in length. In another embodiment, the stem isabout 29 nucleotides in length. Thus, the invention can be practicedusing shRNAs that are synthetically produced, as well as microRNA(miRNA) molecules that are found in nature and can be remodeled tofunction as synthetic silencing short hairpin RNAs.

“RNAi-expressing construct” or “RNAi construct” is a generic term thatincludes nucleic acid preparations designed to achieve an RNAinterference effect. An RNAi-expressing construct comprises an RNAimolecule that can be cleaved in vivo to form an siRNA. For example, anRNAi construct is an expression vector capable of giving rise to ansiRNA in vivo. Exemplary methods of making and delivering long or shortRNAi constructs can be found, for example, in WO01/68836 and WO01/75164.

In certain embodiments of the invention, such as those directed totherapeutic applications, it may be desirable to use siRNAs, includingmodified siRNAs, based on the sequences of the shRNA sequences disclosedherein. For example, it may be desirable to use an siRNA that binds tothe same target sequence as the shRNA sequences disclosed herein. One ofskill in the art can readily design such siRNAs, for example basing thesiRNA sequence on any or all parts of the shRNA sequence that iscomplementary to or binds to the target sequence.

4.4 VECTORS

The vectors described in International application no. PCT/US2008/081193and methods of making and using the vectors are incorporated herein byreference.

shRNAs can be expressed from vectors to provide sustained silencing andhigh yield delivery into almost any cell type. In a certain embodiment,the vector is a viral vector. Exemplary viral vectors includeretroviral, including lentiviral, adenoviral, baculoviral and avianviral vectors. The use of viral vector-based RNAi delivery not onlyallows for stable single-copy genomic integrations but also avoids thenon-sequence specific response via cell-surface toll-like receptor 3(TLR3), which has raised many concerns for the specificity of siRNAmediated effects. In one embodiment of the present invention, a pool ofshRNAs is introduced into murine HSCs using a vector known in the art.

Retroviruses from which the retroviral plasmid vectors can be derivedinclude, but are not limited to, Moloney Murine Leukemia Virus, spleennecrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosisvirus, gibbon ape leukemia virus, human immunodeficiency virus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviralplasmid vector can be employed to transduce packaging cell lines to formproducer cell lines. Examples of packaging cells which can betransfected include, but are not limited to, the PE50l, PA317, R-2,R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectorcan transduce the packaging cells through any means known in the art. Aproducer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding a DNA replication protein. Suchretroviral vector particles then can be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express a DNA replication protein.

In certain embodiments, cells can be engineered using anadeno-associated virus (AAV). AAVs are naturally occurring defectivevimses that require helper viruses to produce infectious particles(Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It isalso one of the few viruses that can integrate its DNA into nondividingcells. Vectors containing as little as 300 base pairs of AAV can bepackaged and can integrate, but space for exogenous DNA is limited toabout 4.5 kb. Methods for producing and using such AAVs are known in theart. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678,5,436,146, 5,474,935, 5,478,745, and 5,589,377. For example, an AAVvector can include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The recombinant AAV vector canbe transfected into packaging cells which are infected with a helpervirus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe polynucleotide construct. These viral particles are then used totransduce eukaryotic cells.

Essentially any method for introducing a nucleic acid construct intocells can be employed. Physical methods of introducing nucleic acidsinclude injection of a solution containing the construct, bombardment byparticles covered by the construct, soaking a cell, tissue sample ororganism in a solution of the nucleic acid, or electroporation of cellmembranes in the presence of the construct. A viral construct packagedinto a viral particle can be used to accomplish both efficientintroduction of an expression construct into the cell and transcriptionof the encoded shRNA. Other methods known in the art for introducingnucleic acids to cells can be used, such as lipid-mediated carriertransport, chemical mediated transport, such as calcium phosphate, andthe like. Thus the shRNA-encoding nucleic acid construct can beintroduced along with components that perform one or more of thefollowing activities: enhance RNA uptake by the cell, promote annealingof the duplex strands, stabilize the annealed strands, or otherwiseincrease inhibition of the target gene.

Expression of endogenous miRNAs is controlled by RNA polymerase II (PolII) promoters. It has been shown that shRNAs are also most efficientlydriven by Pol II promoters, as compared to RNA polymerase III promoters(Dickins et al., 2005, Nat. Genet. 39: 914-921). Therefore, in a certainembodiment, the coding sequence of the RNAi molecule is controlled by aninducible promoter or a conditional expression system, including,without limitation, RNA polymerase type II promoters. Examples of usefulpromoters in the context of the invention are tetracycline-induciblepromoters (including TRE-tight), IPTG-inducible promoters, tetracyclinetransactivator systems, and reverse tetracycline transactivator (rtTA)systems. Constitutive promoters can also be used, as can cell- ortissue-specific promoters. Many promoters will be ubiquitous, such thatthey are expressed in all cell and tissue types. A certain embodimentuses tetracycline-responsive promoters, one of the most effectiveconditional gene expression systems in in vitro and in vivo studies. SeeInternational Patent Application PCT/US2003/030901 (Publication No. WO2004-029219 A2) and Fewell et al., 2006, Drug Discovery Today 11:975-982, for a description of inducible shRNA.

Tetracycline (Tet)-responsive promoters can be used for in vitro and invivo studies. Tet-On is a variation of the Tet-Off system (Gossen andBujard, (1992), Proc. Natl. Acad. Sci. USA, 89:5547-5551), and featuresa modified Tet repressor that has reversed DNA binding properties whencompared to the wild-type Tet-repressor (tetR) encoded in the Tn10Tet-resistance operon of E. coli. The reverse tetracycline-controlledtransactivator (rtTA) is made from a Tet-repressor fused to theactivating domain of virion protein 16 (VP16) of herpes simplex virus(HSV). In contrast to the Tet-Off system, the Tet-On system is optimizedfor induction by the Tet-analogue doxycycline (Dox) only.

Expression of rtTA can be driven by a constitutive promoter of choice.When rtTA is expressed, the presence of Dox leads to a conformationalchange and binding of rtTA-Dox to the Tet operator sequence (tetO) ofthe Tet-resistance operon. The rtTA3 is an improved variant of thereverse tet-transactivator, showing a more sigmoidal induction curve,which is a result of less background activity Off-Dox (tet-On system)and full induction of transgene expression at lower doxycycline (Dox)concentrations (Urlinger et al., (2000), Proc. Natl. Acad. Sci. U.S.A.97, 7963-7968). Seven serial tetO sequences were fused to a minimalcytomegalovirus (CMV) promoter and termed the Tet-responsive element(TRE). The binding of rtTA-Dox, therefore, induces the expression of agene of interest from the minimal CMV promoter. Thus, by placing anshRNA under the control of the TRE, the expression of the RNAi moleculeis inducible by the addition of Dox.

To facilitate the monitoring of the target gene knockdown and theformation and progression of a cancer, cells harboring theRNAi-expressing construct can additionally comprise a marker or reporterconstruct, such as a fluorescent construct. The reporter construct canexpress a marker, such as green fluorescent protein (GFP), enhancedgreen fluorescent protein (EGFP), Renilla Reniformis green fluorescentprotein, GFPmut2, GFPuv4, yellow fluorescent protein (YFP), such asVENUS, enhanced yellow fluorescent protein (EYFP), cyan fluorescentprotein (CFP), enhanced cyan fluorescent protein (ECFP), bluefluorescent protein (BFP), enhanced blue fluorescent protein (EBFP),citrine and red fluorescent protein from discosoma (dsRED). Othersuitable detectable markers include chloramphenicol acetyltransferase(CAT), luminescent proteins such as luciferase lacZ (β-galactosidase)and horseradish peroxidase (HRP), nopaline synthase (NOS), octopinesynthase (OCS), and alkaline phosphatase. The marker gene can beseparately introduced into the cell harboring the shRNA construct (e.g.,co-transfected, etc.). Alternatively, the marker gene can be on theshRNA construct, and the marker gene expression can be controlled by thesame or a separate translation unit, for example, by an IRES (internalribosomal entry site). In a certain embodiment, the marker is a yellowfluorescent protein (VENUS). In some embodiments, the vector can be anRNAi vector (e.g., TRMPV) shown in FIG. 5.

Reporters can also be those that confer resistance to a drug, such asneomycin, ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, doxycycline, and tetracyclin. Reporters can also be lethalgenes, such as herpes simplex virus-thymidine kinase (HSV-TK) sequences,as well as sequences encoding various toxins including the diphtheriatoxin, the tetanus toxin, the cholera toxin and the pertussis toxin. Afurther negative selection marker is the hypoxanthine-guaninephosphoribosyl transferase (HPRT) gene for negative selection in6-thioguanine.

To facilitate the quantification of specific shRNAs in a complexpopulation of cells infected with a library of shRNAs, each shRNAconstruct can additionally comprise a barcode. A barcode is a uniquenucleotide sequence (generally a 19-mer), linked to each shRNA. Thebarcode can be used to monitor the abundance of each shRNA viamicroarray hybridization (Fewell et al., 2006, Drug Discovery Today 11:975-982). In a certain embodiment, each shRNA construct also comprises aunique barcode. For more information on the use of barcodes in shRNApooled analyses, see WO 04/029219, Bernards et al., 2006, Nature Methods3: 701-706, and Chang et al., 2006, Nature Methods 3: 707-714.

The efficacy of RNAi depends on its sequence and on that of its targetsite. In a conventional approach, the potency of RNAi sequences aretested by expressing the molecule and testing the suppression of thetarget mRNA (e.g., QRT-PCR, Northern blot) or its protein product (e.g.,Western blot).

In one embodiment, the expression of a given shRNA from theTet-inducible promoter (or other promoter) can knock-down the cognatereporter-target mRNA construct expressed from an independent promotercloned into the same vector. In this embodiment, cells expressing apotent RNAi lose expression of the fluorescent marker upon induction ofthe Tet-inducible promoter (addition of doxycycline in a Tet-On system)due to RNA interference. Cells expressing a weak RNAi molecule retainexpression of the fluorescent marker due to the lack of a potent RNAiresponse. This differential expression of the fluorescent markerprovides a way of distinguishing RNAi molecules exhibiting varyingknock-down efficacy.

Cells expressing different levels of fluorescence (or no fluorescence)can be gated and sorted by flow cytometry. Potent knockdown cells (nofluorescence) can be differentially isolated from intermediate, weak,and no-knockdown populations. These populations can be expanded suchthat PCR can be performed to clone out the shRNA sequences into otherplasmids, such as bacterial plasmids, which can be used to transformbacteria. In this manner, individual colonies contain a single RNAisequence, and thus, each individual RNAi sequence can be analyzed. Forlarger pools, identification of individual RNAi sequences can beperformed by other methods, such as hybridization on custom arrays.

4.5 RNAI DESIGN AND LIBRARIES

si- and shRNAs targeting genes of interest can be chosen from varioussources. In one embodiment, the RNAi sequences are selected fromexisting libraries. For example, Silva et al. (2005, Nat. Genet. 37:1281-1288), have described extensive libraries of pri-miR-30-basedretroviral expression vectors that can be used to down-regulate almostall known human (at least 28,000) and mouse (at least 25,000) genes.(See RNAi CODEX, a single database that curates publicly available RNAiresources, and provides the most complete access to this growingresource, allowing investigators to see not only released clones butalso those that are soon to be released, available at the Cold SpringHarbor Laboratories website). Pools of shRNAs useful to practice methodsof the invention can be from the “the Cancer 1000” library, which wasconstructed by Steve Elledge and Greg Hannon. The “Cancer 1000” shRNAlibrary includes a mixture of well characterized oncogenes and tumorsuppressor genes in addition to many poorly-characterized genes somehowrelated to cancer, across many ontological groups, as compiled byliterature mining. In another embodiment, the pools of shRNA useful topractice the method of the invention come from a cDNA library thatincludes a mixture of DNA replication genes. A similar library designrationale can be easily applied to construct RNAi libraries targetinggenomes of other organisms, such as the human. Negative controls caninclude shRNAs to genes not present in the organism's genome or emptyvectors.

In another embodiment, si- and shRNAs can be designed de novo. Thesequence coding for an RNAi molecule is referred to as an RNAi codingsequence. The coding sequence can be, for example, a sequence thatencodes an shRNA molecule. Coding sequences for shRNA molecules can bedesigned according to the teachings expressed in Hannon et al. (USPublication No. 2006/0135456), Hannon et al. (International PublicationNo. WO2006/073601), and Dickens et al., (US Publication No.2007/0044164), the contents of which are hereby expressly incorporatedby reference. The choice of the right primary sequence has an importantrole in determining the efficacy and specificity of the resulting RNAiresponse. Current features of design rules for RNAi molecules includethe thermodynamic asymmetry of the RNA duplex, sequence homology of theseed sequence to its cognate target mRNA but not to other mRNAs, and aset of empirical single nucleotide position preferences. Thethermodynamic asymmetry is important since only the strand with the lessstable 5′ end is favorable or exclusively loaded into the RISC and willtherefore serve as the guide strand. The seed sequence comprisesnucleotide positions 2-8 of the guide strand and has been show to be themajor specificity determinant of si- and shRNAs. Single nucleotidepositional preferences include, for example, the A or U at position 10of the guide strand that can promote catalytic RISC-mediated passengerstrand and substrate cleavage.

In certain embodiments, useful interfering RNAs can be designed with anumber of software programs, e.g., the OligoEngine siRNA design tool.Algorithms for in silenco prediction, or algorithms based on anempirically trained neural network, such as BIOPREDsi, can be used.Birmingham et al. (2007, Nat. Protocols 2: 2068-2078) provide acomprehensive overview of prediction algorithms.

In certain embodiments of the invention, such as those directed totherapeutic applications, it may be desirable to use siRNAs, includingmodified siRNAs, based on the sequences of the shRNA sequences disclosedherein. For example, it may be desirable to use an siRNA that binds tothe same target sequence as the shRNA sequences disclosed herein. One ofskill in the art can readily design such siRNAs, for example basing thesiRNA sequence on any or all parts of the shRNA sequence that iscomplementary to or binds to the target sequence.

The siRNAs for use in this invention can have a double-stranded regionof about 16 to 29 base pairs, in particular 19 to 29 base pairs inlength. In a certain embodiment, the duplex region is 21, 22, or 23 basepairs in length. In one aspect, the siRNA comprises a 3′ overhang of 1to 4 nucleotides, in particular 2 nucleotides. Useful siRNAs are highlyspecific for a region of the target gene and can comprise a 19 to 29base pair sequence of the mRNA of a target gene, with at least one, butin particular two or three, base pair mismatches with a non-targetgene-related sequence. In some embodiments, the siRNAs do not bind toRNAs having more than three base pair mismatches with the target region.siRNAs can be prepared extracellularly and intracellularly using avariety of known methods, including chemical synthesis, in vitrotranscription, siRNA expression vectors, and PCR expression cassettes.

Other methods of RNAi can also be used in the practice of thisinvention. See, e.g., International Patent Application PCT/US2003/030901(Publication No. WO 2004-029219 A2) and Fewell et al., 2006, DrugDiscovery Today 11: 975-982, for a description of inducible shRNA, inwhich the vector does not express the shRNA unless a specific reagent isadded. Several studies investigating the function of essential genesusing RNAi rely on inducible shRNA. For example, shRNAmir constructs canbe created based on a tetracycline-responsive promotor system, such thatshRNA expression is regulated by changing doxycycline levels.

In some embodiments, effective RNAi molecules against a target gene maybe designed through the Target Sensor assay, according to the teachingsexpressed in Fellmann et al. (International Publication No.WO2009/055724)(PCT/US08/81193), the contents of which are herebyexpressly incorporated by reference. In further aspects of thisinvention, most potent siRNA or shRNA against a target gene (e.g.,having the most effect in inhibiting cell proliferation or eliminatingcancer cells in vivo) may be identified through use of tet-regulated invivo RNAi screening (Example 6).

4.6 IN VIVO MOUSE MODELS

The methods of the invention use a genetically defined mosaic mousemodel for leukemia, such as the acute myeloid leukemia (AML) model orthe myeloid/lymphoid leukemia (MLL) model. Individual cases of humanleukemia are often associated with certain specific categories ofrecurring genetic abnormalities, such as rearrangements or mutations ofspecific gene loci. For example the MLL gene is fused to a large varietyof more than 50 different partner genes in AML. (Schoch et al., 2003,Blood 102:2395-2402). Some subcategories, for example, 11q23/MLLrearrangements in AML, are associated with poor outcome and resistanceto conventional chemotherapies.

As a mouse model of such human leukemias, we generated mice harboringleukemias with genetic alterations reflecting genetic alterations foundin chemoresistant AML. MLL rearrangements in human AML are commonlyassociated with activating mutations in Nras. To model chemoresistanthuman AML involving MLL translocations, we generated mouse leukemiasco-expressing an MLL gene fusion variant together with oncogenic Nras.We applied a “mosaic” approach involving retroviral transduction ofoncogenes into hematopoietic stem- and progenitor cells, followed byre-transplantation of the genetically-altered cells into syngeneicrecipients (FIG. 12). This strategy enables multiple oncogenes andreporter elements to be introduced in a one-step procedure, therebyfacilitating disease monitoring and reducing the animal husbandryassociated with germline transgenic mice. The resulting primaryleukemias can be isolated and transplanted into secondary recipient miceto assess response to chemotherapy treatment or to evaluate RNAitargets.

In one embodiment, the hematopoetic stem- and progenitor cells are fetalliver cells (FLCs) derived from mouse embryos (e.g., E13.5-15.5embryos). In another embodiment, the hematopoetic stem- and progenitorcells are derived from bone marrow. In one embodiment, an MLL genefusion variant associated with a particularly poor prognosis, MLL/AF9and MLL/ENL (Schoch et al. 2003) can be used, alone or together with anactivated Nras (e.g., Nras^(G12D)). In another embodiment, genealterations associated with less aggressive AML (e.g., AML1/ETO9a) canbe used, alone or together with an activated Nras (e.g., Nras^(G12D)).In a further embodiment, the MLL fusion gene is co-expressed with areporter gene (marker protein) (e.g., enhanced green fluorescent protein(EGFP)) in a bicistronic construct (FIG. 12). In a further embodiment,oncogenic Nras^(G12D) is co-expressed with a marker protein (e.g.,luciferase) that enables imaging of the resulting leukemias bybioluminescence. In a further embodiment, Nras^(G12D) is cloneddownstream of an internal ribosomal entry site (IRES), which reducesexpression of the ectopic Nras gene to near physiological levels (Parikhet al. 2006).

In another embodiment, primary leukemia cells are isolated from diseasedanimals, transplanted in recipient animals and development of leukemiasin the recipient animals is monitored by bioluminescent imaging, usingone or more co-expressed marker proteins (e.g., luciferase co-expressedwith oncogenic Nras^(G12D)).

In another embodiment, a tet transactivator protein (e.g., rtTA3, apotent and non-toxic rtTA variant) together with the oncogeneresponsible for maintaining a malignant phenotype, (e.g. MLL/AF9), isexpressed from one promotor (e.g. LTR) in a bicistronic vector, whereinresulting primary leukemias arising in the animals are comprised of“tet-on competent” leukemia cells.

4.6.1 “Tet-on Competent” shRNA Screening In Vivo

We developed a robust method for in vivo screening using “tet-oncompetent” vectors to identify siRNA molecules with therapeutic efficacyagainst chemoresistant leukemias using transplantable primary leukemiasgenerated through our mosaic mouse model. In particular, we directedthis approach to identify siRNA targets in leukemias involving specificgenetic alterations reflecting those observed in human AML, such asrearrangements of the MLL gene. The tet-on competent vector comprisesthree critical elements that allow for efficient and robust evaluationand/or screening in vivo of shRNAs expressed in transplanted primaryleukemias: (1) inducible expression of shRNA controlled by an inducibletet-responsive promoter, (2) a marker gene, (e.g., dsRed2) co-expressedfrom the same transcript comprising the shRNA allowing monitoring ofshRNA expression, (3) separate selection marker (e.g., Venus=yellowfluorescent protein) expressed from a separate promoter that allows forseparate monitoring of vector integration (e.g., Venus) and shRNAexpression (e.g., dsRed2).

Importantly, we determined that in using the tet-on competent vector toidentify therapeutically potent shRNAs, as a critical prerequisite, itis essential to maintain stable, robust expression of a tettransactivator protein in all transplanted leukemic cells. Otherwise,outgrowth of clones in which shRNA expression is no longer inducible(for example, through selective pressure against expression oftherapeutically active shRNAs) severely compromises success of screeningor evaluation of shRNA activity. As an additional critical element ofthe screening method, we modified our mosaic mouse model byco-expressing a tet transactivator protein (e.g., rtTA3, a potent andnon-toxic rtTA variant) together with the oncogene responsible formaintaining the leukemic phenotype, (e.g. MLL/AF9), from one promotor(e.g. LTR) in a bicistronic vector, ensuring that stable, robust rtTAexpression and inducibility of shRNA expression is maintained in alltransplanted cells, and independently of the identity of the particularshRNA being expressed.

In one embodiment of the invention, primary tet-on competent leukemiacells are isolated from diseased animals, transduced with a “tet-oncompetent” vector and transplanted in recipient animals. In a furtherembodiment, the tet-on competent vector comprises a sequence encoding anshRNA that is complementary to at least a portion of a target gene. Inanother embodiment, primary tet-on competent leukemia cells are isolatedfrom diseased animals and transduced with a plurality of tet-oncompetent vectors, each comprising a sequence encoding a representativeshRNA from an shRNA library, and transduced cells are transplanted intorecipient animals. In a further embodiment, shRNA expression intransplanted cells is monitored by expression of a selection marker(marker protein, reporter gene, e.g., dsRed2) co-expressed from the sametranscript comprising the shRNA. In a further embodiment, integration ofthe tet-on competent vector in transplanted cells is monitored byexpression of a separate selection marker (marker protein, reportergene, e.g., Venus=yellow fluorescent protein) expressed from a separatepromoter that allows for separate monitoring of vector integration(e.g., Venus) and shRNA expression (e.g., dsRed2).

Bicistronic vectors, such as the pIRES2 Vectors from Clonetech (MountainView, Calif.) and the Bicep Vectors from Sigma-Aldrich (St. Louis, Mo.),can be obtained commercially.

Mice harboring a tet-responsive RNA polymerase II promoter can drive thetargeting of a microRNA-based short hairpin RNA to a DNA replicationprotein. rtTA transactivators (“tet-on”) can be activated bytetracycline or its analog doxycycline. Thus, RNA polymerase IIpromoters, including the tet-responsive TRE promoter, can be used toexpress shRNAs complementary to a nucleotide sequence of a target genebased on microRNA precursors. In one embodiment, shRNAs are targeted toa DNA replication protein. Non-limiting example of DNA replicationproteins include replication protein A3 (RPA3), ribonucleotide reductaseM1 (RRM1), cell division cycle 45 (CDC45) and pescadillo 1 (PES1). Incertain embodiments, the shRNA can be or comprise a sequence selectedfrom one of SEQ ID NOs: 8-14. In other embodiments, shRNAs are targetedto a gene encoding a protein shown in Table 1, Table 2, Table 3, orTable 4. Non-limiting examples of specific shRNAs are listed in Tables1, 2, 3, and 4.

4.7 ADDITIONAL METHODS OF SCREENING

The tet-on competent screening systems, along with siRNA targetsidentified herein, are useful for screening candidate therapeutic agentsin vivo, in particular candidate agents capable of enhancing thetherapeutic activity of a particular siRNA in eliminating or otherwiseinhibiting proliferation or survival of tumor cells (e.g., AML and otherleukemias).

The tet-on competent screening systems, in combination with shRNAlibraries, are useful in screening for siRNA targets that enhance thesensitivity of tumor cells (e.g., AML or other leukemias) to achemotherapeutic agent. In one embodiment, the method comprisesintroducing a plurality of transduced into a recipient mouse

In another embodiment, the tet-on competent screening systems, alongwith siRNA targets identified herein, are useful to identify therapeuticagents that modulate downstream substrates or signaling pathwaycomponents of such siRNA targets.

One aspect of the invention is directed to methods for identifyingcompounds that inhibit cell proliferation, and in particularproliferation of tumor cells (e.g., therapy-resistant AML and otherleukemias). In one embodiment, the method comprises (1) introducing intoa cancer cell an RNAi molecule that is complementary to a nucleotidesequence of a target gene, wherein the target gene encodes a DNAreplication protein or an epigenetic modifier (e.g., an enzyme thatmodifies chromatin); (2) contacting the cancer cell with a candidatecompound; and (3) determining whether the candidate compound inhibitscell proliferation. In another embodiment, the method further comprises:administering an effective amount of the compound to a non-human animalhaving a tumor; and monitoring tumor growth in the non-human animal. Incertain embodiments, the method further comprises comparing tumor growthin the non-human animal treated with the compound to tumor growth in thenon-human animal not treated with the compound. Monitoring tumor growthcan be carried out according to methods known in the art and cancomprise evaluating the size of the cells comprising the tumor,measuring cell viability, or a carrying out a combination of monitoringmodes described.

A further aspect of the invention is a method for testing a cancerarising from an Nras^(G12D)/shRNA tumor oncogene-transformed cancer forsensitivity to a treatment. Another aspect of this invention is a methodfor testing a chemotherapy-resistant AML (e.g., human AML harboring anMLL-fusion oncogene, AML arising from hematopoietic stem- and progenitorcells transduced with an MLL-fusion+oncogenic Nras, see e.g., Example6). Cancer cells, such as leukemia cells, can be cultured in vitro. Thecells are subsequently contacted with a candidate treatment andmonitored for growth (e.g., by observing cell number, confluence inflasks, staining to distinguish viable from nonviable cells). Failure toincrease in viable cell number, slower rate of increase in cell number,or a decline in viable cell number, compared to cells which areuntreated or mock-treated, is an indication of sensitivity to thetreatment. The treatment to be tested can be one or more substances, forexample, those compounds identified in the screen of the invention, aknown anti-cancer agent (e.g., a chemotherapy drug), such as adriamycin,cylophosphamide, prednisone, vincristine, or a radioactive source. Thetreatment can also be exposure to various kinds of energy or particles,such as gamma-irradiation, or can be a combination of approaches. Insome cases, the treatment can also be administration of one or moresubstances or exposure to conditions, or a combination of both, whereinthe effects of the treatment as anti-cancer therapy are unknown. Inaddition, candidate compounds can be selected based on their effect onproteins that bind or interact with DNA replication proteins.

Candidate compounds can be further tested in leukemias orleukemia-related tumors in situ in a mouse. Animals can be testedessentially as described in U.S. Pat. No. 6,583,333.

A compound that inhibits cell proliferation can be a small molecule thatbinds to and disrupts or inhibits the function of DNA replicationproteins. A compound that inhibits an epigenetic pathway can be a smallmolecule that binds to and disrupts or inhibits the enzymatic functionof an epigenetic modifier (e.g., an enzyme that modifies chromatin).Small molecules are a diverse group of synthetic and natural substancesgenerally having low molecular weights. They can be isolated fromnatural sources (for example, plants, fungi, microbes and the like),obtained commercially and/or available as libraries or collections, orsynthesized. Candidate small molecules that disrupt the function of DNAreplication proteins or epigenetic modifiers can be identified via insilico screening or high-through-put (HTP) screening of combinatoriallibraries. Most conventional pharmaceuticals, such as aspirin,penicillin, and many chemotherapeutics, are small molecules, and can beobtained commercially, can be chemically synthesized, or can be obtainedfrom random or combinatorial libraries as described below (Werner etal., (2006) Brief Funct. Genomic Proteomic 5(1):32-6).

Knowledge of the primary sequence of a molecule of interest, such as DNAreplication proteins or epigenetic modifiers discussed herein, canprovide information as to the inhibitors or antagonists of the proteinof interest in addition to agonists. Identification and screening ofagonists and antagonists is further facilitated by determiningstructural features of the protein, e.g., using X-ray crystallography,neutron diffraction, nuclear magnetic resonance spectrometry, and othertechniques for structure determination. These techniques provide for therational design or identification of agonists and antagonists.

Test compounds, such as cell proliferation inhibitors (e.g., inhibitorsof DNA replication proteins) or epigenetic modifiers (e.g., enzymes thatmodify chromatin), can be screened from large libraries of synthetic ornatural compounds (see Wang et al., (2007) Curr Med Chem, 14(2):133-55;Mannhold (2006) Curr Top Med Chem, 6 (10):1031-47; and Hensen (2006)Curr Med Chem 13(4):361-76). Numerous means are currently used forrandom and directed synthesis of saccharide, peptide, and nucleic acidbased compounds. Synthetic compound libraries are commercially availablefrom Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex(Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource(New Milford, Conn.). A rare chemical library is available from Aldrich(Milwaukee, Wis.). Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available, e.g.from Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or arereadily producible. Additionally, natural and synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical, and biochemical means (Blondelle et al., (1996) TibTech 14:60).

Methods for preparing libraries of molecules are well known in the artand many libraries are commercially available. Libraries of interest inthe invention include peptide libraries, randomized oligonucleotidelibraries, synthetic organic combinatorial libraries, and the like.Degenerate peptide libraries can be readily prepared in solution, inimmobilized form as bacterial flagella peptide display libraries or asphage display libraries. Peptide ligands can be selected fromcombinatorial libraries of peptides containing at least one amino acid.Libraries can be synthesized of peptoids and non-peptide syntheticmoieties. Such libraries can further be synthesized to containnon-peptide synthetic moieties, which are less subject to enzymaticdegradation compared to their naturally-occurring counterparts.Libraries are also meant to include, for example, but are not limitedto, peptide-on-plasmid libraries, polysome libraries, aptamer libraries,synthetic peptide libraries, synthetic small molecule libraries,neurotransmitter libraries, and chemical libraries. The libraries canalso comprise cyclic carbon or heterocyclic structure and/or aromatic orpolyaromatic structures substituted with one or more of the functionalgroups described herein.

Small molecule combinatorial libraries can also be generated andscreened. A combinatorial library of small organic compounds is acollection of closely related analogs that differ from each other in oneor more points of diversity and are synthesized by organic techniquesusing multi-step processes. Combinatorial libraries include a vastnumber of small organic compounds. One type of combinatorial library isprepared by means of parallel synthesis methods to produce a compoundarray. A compound array can be a collection of compounds identifiable bytheir spatial addresses in Cartesian coordinates and arranged such thateach compound has a common molecular core and one or more variablestructural diversity elements. The compounds in such a compound arrayare produced in parallel in separate reaction vessels, with eachcompound identified and tracked by its spatial address. Examples ofparallel synthesis mixtures and parallel synthesis methods are providedin U.S. Ser. No. 08/177,497, filed Jan. 5, 1994 and its correspondingpublished PCT application, WO95/18972, and in U.S. Pat. No. 5,712,171 gand its corresponding published PCT application, WO96/22529, which arehereby incorporated by reference.

Examples of chemically synthesized libraries are described in Fodor etal., (1991) Science 251:767-773; Houghten et al., (1991) Nature354:84-86; Lam et al., (1991) Nature 354:82-84; Medynski, (1994)BioTechnology 12:709-710; Gallop et al., (1994) J. Medicinal Chemistry37(9):1233-1251; Ohlmeyer et al., (1993) Proc. Natl. Acad. Sci. USA90:10922-10926; Erb et al., (1994) Proc. Natl. Acad. Sci. USA91:11422-11426; Houghten et al., (1992) Biotechniques 13:412;Jayawickreme et al., (1994) Proc. Natl. Acad. Sci. USA 91:1614-1618;Salmon et al., (1993) Proc. Natl. Acad. Sci. USA 90:11708-11712; PCTPublication No. WO 93/20242, dated Oct. 14, 1993; and Brenner et al.,(1992) Proc. Natl. Acad. Sci. USA 89:5381-5383.

In one non-limiting example, non-peptide libraries, such as abenzodiazepine library (see e.g., Bunin et al., (1994) Proc. Natl. Acad.Sci. USA 91:4708-4712), can be screened. Peptoid libraries, such as thatdescribed by Simon et al., (1992) Proc. Natl. Acad. Sci. USA89:9367-9371, can also be used. Another example of a library that can beused, in which the amide functionalities in peptides have beenpermethylated to generate a chemically transformed combinatoriallibrary, is described by Ostresh et al. (1994), Proc. Natl. Acad. Sci.USA 91:11138-11142.

Screening the libraries can be accomplished by any variety of commonlyknown methods. See, for example, the following references, whichdisclose screening of peptide libraries: Parmley and Smith, (1989) Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, (1990) Science249:386-390; Fowlkes et al., (1992) BioTechniques 13:422-427; Oldenburget al., (1992) Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al.,(1994) Cell 76:933-945; Staudt et al., (1988) Science 241:577-580; Bocket al., (1992) Nature 355:564-566; Tuerk et al., (1992) Proc. Natl.Acad. Sci. USA 89:6988-6992; Ellington et al., (1992) Nature355:850-852; U.S. Pat. Nos. 5,096,815; 5,223,409; and 5,198,346, all toLadner et al.; Rebar et al., (1993) Science 263:671-673; and PCT Pub. WO94/18318.

4.8 METHODS OF TREATMENT

Another aspect of the invention is a method of treating cancer byinhibiting cancer cell proliferation through the downregulation of atarget gene identified through the methods of the invention as a genewhose expression is necessary for survival of the cancer cell. In someaspects, the target gene encodes a DNA replication protein. In otheraspects, the target gene encodes an epigenetic modifier. The proteinencoded by the target gene can be downregulated directly, by introducinginto a cancer cell an RNAi molecule against the DNA replication protein,or indirectly, by targeting an upstream factor that regulatesexpression, inhibition or activation of the target protein. Theinvention provides methods of treating a cancer in a subject byintroducing into cells a pharmaceutical composition comprising atherapeutic agent, such therapeutic agent comprising an shRNA expressionconstruct, an siRNA or another RNAi molecule. In certain embodiments ofthe invention, such as those directed to therapeutic applications, itmay be desirable to use siRNAs, including modified siRNAs, based on thesequences of the shRNA sequences disclosed herein. For example, it maybe desirable to use an siRNA that binds to the same target sequence asthe shRNA sequences disclosed herein. One of skill in the art canreadily design such siRNAs, for example basing the siRNA sequence on anyor all parts of the shRNA sequence that is complementary to or binds tothe target sequence.

For example, the shRNA can be reliably expressed in vivo in a variety ofcell types. In certain embodiments the cells are administered in orderto treat a condition. There are a variety of mechanisms by which shRNAexpressing cells can be useful for treating cancer. For example, acondition can be caused, in part, by a population of cells expressing anundesirable gene. These cells can be ablated and replaced withadministered cells comprising shRNA that decreases expression of theundesirable gene. An shRNA can be targeted to essentially any gene, thedecreased expression of which can be helpful in treating cancer.

In one embodiment, the invention can be a method for treating cancercomprising identifying in a cancer cell increased expression or copynumber of at least one gene that encodes a DNA replication protein orepigenetic modifier as compared with that in a normal cell andinhibiting the DNA replication protein or epigenetic modifier.Non-limiting examples of DNA replication proteins include replicationprotein A3 (RPA3), ribonucleotide reductase M1 (RRM1), cell divisioncycle 45 (CDC45) and pescadillo 1 (PES1). shRNAs that are complementaryto a nucleotide sequence of a gene encoding a protein shown in Table 1provides additional examples of DNA replication proteins. Non-limitingexamples of epigenetic modifiers include AOF2, EED, HDAC, MEN1, SMARCA4,SMARCD1, SUZ12 and WHSC111.

In another embodiment, the invention can be a method for inhibitingproliferation of a cancer cell by introducing into the cancer cell asmall interfering RNA (siRNA) comprising a nucleic acid sequence that iscomplementary to a nucleotide sequence of a target gene. The target geneencodes a DNA replication protein, wherein the DNA replication proteinis selected from the group consisting of replication protein A3 (RPA3),ribonucleotide reductase M1 (RRM1), cell division cycle 45 (CDC45), andpescadillo 1 (PES1). In another aspect, the target gene encodes anepigenetic modifier, wherein the epigenetic modifier is selected fromthe group consisting of AOF2, EED, HDAC, MEN1, SMARCA4, SMARCD1, SUZ12and WHSC111.

In a further embodiment, the invention can be a method for inhibitingproliferation of a cancer cell by introducing into the cancer cell anexpression vector comprising a nucleic acid sequence encoding a shorthairpin RNA (shRNA) operably linked to a RNA polymerase promoter. TheshRNA comprises a loop and a duplex region, wherein the duplex regioncomprises a sequence that is complementary to a nucleotide sequence of atarget gene. The target gene encodes a DNA replication protein, whereinthe DNA replication protein is selected from the group consisting ofreplication protein A3 (RPA3), ribonucleotide reductase M1 (RRM1), celldivision cycle 45 (CDC45) and pescadillo 1 (PES1) thereby inhibitingproliferation of a cancer cell upon expression of the shRNA. In anotheraspect, the target gene encodes an epigenetic modifier, wherein theepigenetic modifier is selected from the group consisting of AOF2, EED,HDAC, MEN1, SMARCA4, SMARCD1, SUZ12 and WHSC111, thereby inhibitingproliferation of a cancer cell upon expression of the shRNA.

In some embodiments, the methods for treating cancer and for inhibitingproliferation of a cancer cell can further comprise administering achemotherapy drug to the cancer cell. Cytotoxic drugs (for example,chemotherapy drugs) that interfere with critical cellular processesincluding DNA, RNA, and protein synthesis, can also be administered incombination with the introduction of an RNAi molecule. Some non-limitingexamples of chemotherapy drugs include: aminoglutethimide, amsacrine,asparaginase, bcg, anastrozole, bleomycin, buserelin, bicalutamide,busulfan, capecitabine, carboplatin, camptothecin, chlorambucil,cisplatin, carmustine, cladribine, colchicine, cyclophosphamide,cytarabine, dacarbazine, cyproterone, clodronate, daunorubicin,diethylstilbestrol, docetaxel, dactinomycin, doxorubicin, dienestrol,etoposide, exemestane, filgrastim, fluorouracil, fludarabine,fludrocortisone, epirubicin, estradiol, gemcitabine, genistein,estramustine, fluoxymesterone, flutamide, goserelin, leuprolide,hydroxyurea, idarubicin, levamisole, imatinib, lomustine, ifosfamide,megestrol, melphalan, interferon, irinotecan, letrozole, leucovorin,ironotecan, mitoxantrone, nilutamide, medroxyprogesterone,mechlorethamine, mercaptopurine, mitotane, nocodazole, octreotide,methotrexate, mitomycin, paclitaxel, oxaliplatin, temozolomide,pentostatin, plicamycin, suramin, tamoxifen, porfimer, mesna,pamidronate, streptozocin, teniposide, procarbazine, titanocenedichloride, raltitrexed, rituximab, testosterone, thioguanine,vincristine, vindesine, thiotepa, topotecan, tretinoin, vinblastine,trastuzumab, and vinorelbine.

In one embodiment, the chemotherapy drug is an alkylating agent, anitrosourea, an anti-metabolite, a topoisomerase inhibitor, a mitoticinhibitor, an anthracycline, a corticosteroid hormone, or a sex hormone.

An alkylating agent works directly on DNA to prevent the cancer cellfrom propagating. These agents are not specific to any particular phaseof the cell cycle. In one embodiment, alkylating agents can be selectedfrom busulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide,ifosfamide, dacarbazine (DTIC), mechlorethamine (nitrogen mustard),melphalan, and temozolomide.

An antimetabolite makes up the class of drugs that interfere with DNAand RNA synthesis. These agents work during the S phase of the cellcycle and are commonly used to treat leukemias, tumors of the breast,ovary, and the gastrointestinal tract, as well as other cancers. In oneembodiment, an antimetabolite can be 5-fluorouracil, capecitabine,6-mercaptopurine, methotrexate, gemcitabine, cytarabine (ara-C),fludarabine, or pemetrexed.

Topoisomerase inhibitors are drugs that interfere with the topoisomeraseenzymes that are important in DNA replication. Some examples oftopoisomerase I inhibitors include topotecan and irinotecan while somerepresentative examples of topoisomerase II inhibitors include etoposide(VP-16) and teniposide.

Anthracyclines are chemotherapy drugs that also interfere with enzymesinvolved in DNA replication. These agents work in all phases of the cellcycle and thus, are widely used as a treatment for a variety of cancers.In one embodiment, an anthracycline used with respect to the inventioncan be daunorubicin, doxorubicin (Adriamycin), epirubicin, idarubicin,or mitoxantrone.

Non-limiting examples of cancers that can be treated according to themethods of the invention include, for example, leukemias, acutelymphocytic leukemia, myeloid/lymphoid or mixed lineage leukemia (MLL),chronic lymphocytic leukemia, lymphomas, B cell lymphoma, T celllymphoma, non-Hodgkins lymphoma, Hodgkins lymphoma, myeloma,hematopoietic neoplasias, thymoma, lymphoma, sarcoma, lung cancer, livercancer, colon cancer, renal cancer, breast cancer, bladder cancer,uterine cancer, cervical cancer and other soft tissue and solid tumorcancers, and including chemotherapy-resistant and radiationtherapy-resistant cancers.

The cancer cell can be a human cancer cell. The cancer cell can also bea mammalian cancer cell including, but not limited to, a non-primatecancer cell (e.g., a cow, pig, bird, sheep, goat, horse, cat, dog, rat,and mouse) and a primate cancer cell (e.g., a monkey, such as acynomolgous monkey, a chimpanzee, and a human). The cancer cell can alsobe a non-human cancer cell, such as from a bird (e.g., a quail, chicken,or turkey), a farm animal (e.g., a cow, horse, pig, or sheep), a pet(e.g., a cat, dog, or guinea pig), or laboratory animal (e.g., an animalmodel for a disorder or disease).

4.9 PHARMACEUTICAL COMPOSITIONS AND ADMINISTRATION

Another aspect of the invention is a pharmaceutical compositioncomprising a therapeutic agent for the treatment of cancer (for example,leukemias such as AML or MLL). The composition has specific utility totreat a cancer by targeting a gene identified through the methods of theinvention as a gene whose expression is necessary for survival of thecancer cell. Such a gene can be a gene encoding a DNA replicationprotein described herein. In one aspect, the pharmaceutical compositioncan be used for the treatment of cancer in which the activity orexpression of a protein encoded by the target gene is greater in thecancer cell than in normal tissue. An embodiment of this invention canbe practiced using inhibitors directed to the DNA replication proteinslisted in Table 1. A further embodiment of this invention can bepracticed using inhibitors directed to epigenetic modifier proteinsidentified through the methods of this invention as novel therapeutictargets for chemotherapy-resistant AML, in particular Eed, Suz12, Aof2,Smarca4, Smarcd1, Men1, Hdac3, and Whs111.

In one aspect of this invention, inhibitors directed to the identifiedtherapeutic targets described herein, including compounds that inhibitor disrupt the function of the target gene and also siRNAs or modifiedsiRNAs, are administered in therapeutically effective amounts. Intreating disease, a therapeutically effective amount (“effectiveamount”) is a dose administered to a patient that is sufficient toprovide a medically desirable result. For example, a therapeuticallyeffective amount is an amount that inhibits or halts the progression ofthe particular disease, for example, progression of a chemotherapyresistant cancer. In treating cancer, an effective amount of aninhibitor is for example, an amount necessary to restore sensitivity toanother chemotherapeutic agent, inhibit cancer cell replication,increase apoptosis in cancer cells, regress the cancer, reduce cancercell load, suppress further growth of the cancer or reduce one or moresigns or symptoms of the cancer. In some embodiments, inhibitorsdirected to the identified therapeutic targets described herein areadminstered as a combination drug therapy. In some aspects, combinationtherapy will include administration of the inhibitor in combination withan existing chemotherapy drug.

Methods of the invention provide for the administration of an RNAimolecule or for compounds identified in the screening methods above thatinhibit cellular proliferation or eliminate cancer cells in vivo. Insome embodiments, a method comprises introducing into a subject atransfected stem cell comprising a siRNA or a nucleic acid constructencoding an shRNA. The siRNA or the shRNA is complementary to at least aportion of a target gene, wherein the transfected stem cell exhibitsdecreased expression of the target gene, and wherein the transfectedstem cell gives rise to a transfected tumor cell in vivo. The targetgene can encode a DNA replication protein, wherein the DNA replicationprotein is selected from the group consisting of replication protein A3(RPA3), ribonucleotide reductase M1 (RRM1), cell division cycle 45(CDC45) and pescadillo 1 (PES1) thereby inhibiting proliferation of acancer cell. In another aspect, the target gene encodes an epigeneticmodifier, wherein the epigenetic modifier is selected from the groupconsisting of AOF2, EED, HDAC, MEN1, SMARCA4, SMARCD1, SUZ12 andWHSC111. For example, the stem cell can be derived from an animal thathas a genetic predisposition to tumorigenesis, such as an oncogeneover-expressing animal (e.g. E-myc mice) or a tumor suppressor knockout(e.g., p53−/− animal). Alternatively, an animal comprising the stemcells can be exposed to carcinogenic conditions such that tumorscomprising cells derived from the stem cells are generated. An animalhaving tumors can be treated with a chemotherapeutic or other anti-tumorregimen, and the effect of this regimen on cells expressing the shRNAcan be evaluated.

In certain embodiments, the invention provides a composition formulatedfor administration to a subject, such as a human or veterinary subject.A composition so formulated can comprise a stem cell comprising anucleic acid construct encoding an shRNA designed to decrease theexpression of a target gene. A composition can also comprise apharmaceutically acceptable excipient. Any suitable cell can be used.For example, cells to be transfected can be essentially any type of cellfor implantation into in a subject. The cell having the target gene canbe germ line or somatic, totipotent or pluripotent, dividing ornon-dividing, parenchymal or epithelial, immortalized or transformed, orthe like. The cell can be a stem cell or a differentiated cell. Aftertransfection, stem cells can be administered to a subject, or culturedto form differentiated stem cells (e.g., embryonic stem cells culturedto form neural, hematopoietic or pancreatic stem cells) or cultured toform differentiated cells.

Stem cells can be stem cells recently obtained from a donor, and incertain embodiments, the stem cells are autologous stem cells. Stemcells can also be from an established stem cell line that is propagatedin vitro. Suitable stem cells include embryonic stems and adult stemcells, whether totipotent, pluripotent, multipotent or of lesserdevelopmental capacity. Stem cells can be derived from mammals, such asrodents (e.g. mouse or rat), primates (e.g. monkeys, chimpanzees orhumans), pigs, or ruminants (e.g. cows, sheep and goats). Examples ofmouse embryonic stem cells include: the JM1 ES cell line described in M.Qiu et al., Genes Dev 9, 2523 (1995), and the ROSA line described in G.Friedrich, P. Soriano, Genes Dev 5, 1513 (1991), and mouse ES cellsdescribed in U.S. Pat. No. 6,190,910. Many other mouse ES lines areavailable from Jackson Laboratories (Bar Harbor, Me.). Examples of humanembryonic stem cells include those available through the followingsuppliers: Arcos Bioscience, Inc., Foster City, Calif.; CyThera, Inc.,San Diego, Calif.; BresaGen, Inc., Athens, Ga.; ES Cell International,Melbourne, Australia; Geron Corporation, Menlo Park, Calif.; Universityof California, San Francisco, Calif.; and Wisconsin Alumni ResearchFoundation, Madison, Wis. In addition, examples of embryonic stem cellsare described in the following U.S. patents and published patentapplications: U.S. Pat. Nos. 6,245,566; 6,200,806; 6,090,622; 6,331,406;6,090,622; 5,843,780; 20020045259; 20020068045. In certain embodiments,the human ES cells are selected from the list of approved cell linesprovided by the National Institutes of Health. Examples of human adultstem cells include those described in the following U.S. patents andpatent applications: U.S. Pat. Nos. 5,486,359; 5,766,948; 5,789,246;5,914,108; 5,928,947; 5,958,767; 5,968,829; 6,129,911; 6,184,035;6,242,252; 6,265,175; 6,387,367; 20020016002; 20020076400; 20020098584;and, for example, in PCT publication WO 01/11011. In certainembodiments, a suitable stem cell can be derived from a cell fusion ordedifferentiation process, such as described in U.S. patent application20020090722, in PCT publications WO 02/38741, WO 01/51611, WO 99/63061,and WO 96/07732.

In some embodiments, stem cells can be hematopoietic or mesenchymal stemcells, such as stem cell populations derived from adult human bonemarrow. Recent studies suggest that marrow-derived hematopoietic (HSCs)and mesenchymal stem cells (MSCs), which are readily isolated, have abroader differentiation potential than previously recognized. Manypurified HSCs not only give rise to all cells in blood, but can alsodevelop into cells normally derived from endoderm, like hepatocytes(Krause et al., 2001, Cell 105: 369-77; Lagasse et al., 2000 Nat Med 6:1229-34). In at least one report (Lagasse et al, 2000 Nat Med 6:1229-34), the possibility of somatic cell fusion was ruled out. MSCsappear to be similarly multipotent, producing progeny that can, forexample, express neural cell markers (Pittenger et al., 1999 Science284: 143-7; Zhao et al., 2002 Exp Neural 174: 11-20).

In other embodiments, stem cells are derived from an autologous sourceor an HLA-type matched source. For example, HSCs can be obtained fromthe bone marrow of a subject in need of ex vivo cell therapy andcultured by a method described herein to generate an autologous cellcomposition. Other sources of stem cells are easily obtained from asubject, such as stem cells from muscle tissue, stem cells from skin(dermis or epidermis) and stem cells from fat. Stem cell compositionscan also be derived from banked stem cell sources, such as bankedamniotic epithelial stem cells or banked umbilical cord blood cells.

Transfected cells can also be used in the manufacture of a medicamentfor the treatment of subjects. Examples of pharmaceutically acceptableexcipients include matrices, scaffolds, or other substrates to whichcells can attach (optionally formed as solid or hollow beads, tubes, ormembranes), as well as reagents that are useful in facilitatingadministration (e.g. buffers and salts), preserving the cells (e.g.chelators such as sorbates, EDTA, EGTA, or quaternary amines or otherantibiotics), or promoting engraftment. Cells can be encapsulated in amembrane or in a microcapsule. Cells can be placed in microcapsulescomposed of alginate or polyacrylates. (Lim et al. (1980) Science210:908; O'Shea et al. (1984) Biochim. Biochys. Acta. 840:133; Sugamoriet at (1989) Trans. Am. Soc. Artif. Intern. Organs 35:791; Levesque etal. (1992) Endocrinology 130:644; and Lim et al. (1992) Transplantation53:1180).

Additional methods for encapsulating cells are known in the art.(Aebischer et al. U.S. Pat. No. 4,892,538; Aebischer et al. U.S. Pat.No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaegeret al. (1990) Prog. Brain Res. 82:4146; and Aebischer et al. (1991) J.Biomech. Eng. 113:178-183, U.S. Pat. No. 4,391,909; U.S. Pat. No.4,353,888; Sugamori et al. (1989) Trans. Am. Artif. Intern. Organs35:791-799; Sefton et al. (1987) Biotehnol. Bioeng. 29:1135-1143; andAebischer et al. (1991) Biomaterials 12:50-55).

The site of implantation of cell compositions can be selected by one ofskill in the art depending on the type of cell and the therapeuticobjective. Exemplary implantation sites include intravenous orintraarterial administration, administration to the liver (via portalvein injection), the peritoneal cavity, the kidney capsule or the bonemarrow.

In other embodiments, the invention provides a composition formulatedfor administration to a subject, such as a human or veterinary subject,comprising an RNAi molecule identified according to the screeningmethods described above. The RNAi molecule or the candidate compoundidentified according to the screening methods described above can beadministered to a human or non-human animal, for example, a veterinarysubject such as a non-human primate, a rodent (e.g., a mouse or rat), alagomorph (e.g., a rabbit), a canid (e.g. a domestic dog), a feline(e.g., a domestic cat), an equine (e.g., a horse), or a bovine, (e.g., acow). In general, animals with complete or near complete genome projectsare useful.

Modification and delivery of RNAi molecules in vivo can be accomplishedas described in U.S. Pat. Nos. 6,627,616, 6,897,068, 6,379,966; in U.S.Patent Application Publication Nos. US 2007/0281900 (Dec. 6, 2007) andUS 2007/0293449 (Dec. 20, 2007); and in Vorhies and Nemunaitis J J,Methods Mol Biol. 2009; 480:11-29, López-Fraga M et al., Infect DisordDrug Targets. 2008 December; 8(4):262-73, Watts et al., Drug DiscovToday. 2008 October; 13(19-20):842-55, Lu and Woodle, Methods Mol Biol.2008; 437:93-107, de Fougerolles, Hum Gene Ther. 2008 February;19(2):125-32, Rossi J J, Hum Gene Ther. 2008 April; 19(4):313-7, BeltingM and Wittrup A. Methods Mol Biol. 2009; 480:1-10, Pushparaj et al., JDENT RES 2008; 87: 992-1003, Shrivastava and Srivastava, Biotechnol J.2008 March; 3(3):339-53, and Raemdonck K, et al., Drug Discov Today.2008 November; 13(21-22):917-31, Castanotto D & Rossi J J, Nature 2009January; 457:426-433, Davis M et al., Nature advance online publication(21 Mar. 2010) doi:10.1038/nature08956, each of which are incorporatedby reference in their entireties.

The candidate compound identified according to the screening methodsdescribed above can be administered by subcutaneous, intra-muscular,intra-peritoneal, or intravenous injection; infusion; oral, nasal, ortopical delivery; or a combination thereof.

Compounds can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions can comprise a compound identifiedin further screens described above (for example, a cell proliferationinhibitor such as a compound that disrupts the function of a DNAreplication protein) and a pharmaceutically acceptable carrier.

According to the invention, a pharmaceutically acceptable carrier cancomprise any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Any conventional media or agent that is compatible with theactive compound can be used. Supplementary active compounds can also beincorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, Cremophor™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, a pharmaceutically acceptable polyol like glycerol,propylene glycol, liquid polyethylene glycol, and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it can be useful to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention.

All publications and other references mentioned herein are incorporatedby reference in their entirety, as if each individual publication orreference were specifically and individually indicated to beincorporated by reference. Publications and references cited herein arenot admitted to be prior art.

5. EXAMPLES OF THE INVENTION

Examples are provided below to facilitate a more complete understandingof the invention. The following examples illustrate the exemplary modesof making and practicing the invention. However, the scope of theinvention is not limited to specific embodiments disclosed in theseExamples, which are for purposes of illustration only, since alternativemethods can be utilized to obtain similar results.

5.1 Example 1 In Vitro Screening Assay for RNAi Target Identification

5.1.1 Construction of pLM-Puro Plasmid and Subcloning of shRNAs

The mir30-based shRNAs used in this invention are most effective inknocking down expression of their target gene if they are under thetranscriptional control of a Pol II promoter. shRNAs, with the exceptionof EBNA1mi1666, Lucifmi203, ORC1mi550, ORC2mi1903, and ORC2mi1981, werepurchased from Open Biosystems, which provided the shRNAs in the pSM2plasmid, containing a Pol III promoter (U6 promoter). All shRNAs weresub-cloned from pSM2 to the pLM-puro plasmid, where shRNA expression isregulated by an upstream LTR element that functions as a Pol IIpromoter.

The pLM-puro plasmid was constructed as follows. The pMSCV plasmid(obtained from Clontech) was linearized with EcoR1 (unique restrictionsite in the multiple cloning site of pMSCV) and the following shortduplex was ligated into pMSCV:

(SEQ ID NO: 15) aattcgtaaaacgcgtacgacggcct (SEQ ID NO: 16)gcatttt gcgcatgctgccgga ttaa

This insert encodes a single MluI restriction site and has sticky endscompatible for ligation into the pMSCV plasmid. The result is theaddition of a unique MluI restriction site into the multiple cloningsite of pMSCV, which was named pMSCV-MluI. A functional EcoRIrestriction site in pMSCV-MluI is retained at the 5′ end of the insert,however the EcoRI site at the 3′ end of the insert is renderednon-functional by the substitution of the C in GAATTC with an A to yieldGAATTA.

Next, construction of pLM-puro was completed by sub-cloning theSalI-MfeI fragment from the pSM2 plasmid into the compatible XhoI/EcoR1sites in the multiple cloning site of pMSCV-MluI. The Sal1-MfeI fragmentfrom pSM2 encodes the 5′ Mir30 flanking sequence, cloning site forinserting the shRNA sequence, and the 3′ Mir30 flanking sequence. Thisligation results in loss of the XhoI and EcoRI sites from the multiplecloning site of pMSCV-MluI. The pLM-puro plasmid derives from its pMSCVbackbone functional LTR sequences required for expression of theinserted shRNA, as well as sequences necessary for packaging of theshRNA expression construct into viral particles formed aftertransfection of the plasmid into a packaging cell line. The pLM-puroplasmid also derives from its pMSCV backbone expression of a puromycinresistance marker that enables selection of human cells stablytransduced with the shRNA expression cassette.

shRNAs were subcloned from pSM2 into pLM-puro by digesting both plasmidswith XhoI and MluI. This resulted in a fragment encoding the shRNA, 3′Mir30 flanking sequence, and a short DNA sequence that is unique incomposition for each shRNA and termed a barcode. The barcode can be usedin array-based high throughput assay systems. This fragment was thenligated into the unique XhoI site of pLM-puro that is just downstream ofthe 5′ Mir30 flanking sequence and the unique MluI site in pLM-purodownstream of the 3′ Mir30 flanking sequence.

5.1.2 Preparation of pLM-Puro Plasmids Encoding shRNAs not in pSM2

The shRNAs against ORC1, ORC2, EBNA1, and Ff-luciferase used in thiswork were generated as follows. The following 97-mer oligonucleotideswere purchased:

ORC1mi550:  (SEQ ID NO: 17)TGCTGTTGACAGTGAGCGCGGAAATATTCTGGTATGATTATAGTGAAGCCACAGATGTATAATCATACCAGAATATTTCCTTGCCTACTGCCTCGGA ORC2mi1903: (SEQ ID NO: 18) TGCTGTTGACAGTGAGCGAGGAACTGATGGAGTAGAGTATTAGTGAAGCCACAGATGTAATACTCTACTCCATCAGTTCCCTGCCTACTGCCTCGGA  ORC2mi1981: (SEQ ID NO: 19) TGCTGTTGACAGTGAGCGAGAGGCTTGAAGCTTTCCTTTATAGTGAAGCCACAGATGTATAAAGGAAAGCTTCAAGCCTCCTGCCTACTGCCTCGGA EBNA1mi1666: (SEQ ID NO: 20) TGCTGTTGACAGTGAGCGCGTCCATTGTCTGTTATTTCATTAGTGAAGCCACAGATGTAATGAAATAACAGACAATGGACTTGCCTACTGCCTCGGA Lucifmi203: (SEQ ID NO: 21) TGCTGTTGACAGTGAGCGCCGATATGGGCTGAATACAAATTAGTGAAGCCACAGATGTAATTTGTATTCAGCCCATATCGTTGCCTACTGCCTCGGA

A PCR amplification was then performed using the above 97mers astemplates and the following oligonucleotides as primers:

5′ Primer:  (SEQ ID NO: 22) CAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGTGAGCG 3′Primer:  (SEQ ID NO: 23) CTAAAGTAGCCCCTTGAATTCCGAGGCAGTAGGCA

The resulting PCR product was then digested with XhoI and EcoRI andinserted into the unique XhoI and EcoRI sites of pLM-puro locatedbetween the 5′ Mir30 and 3′Mir30 flanking sequences. These plasmids donot encode unique barcode sequences for the respective shRNAs.

The sequence of the inserts for each shRNA are as follows. Thenucleotides encoding the shRNA sequences that target each respectivemRNA for destruction are underlined.

ORC1mi550:  (SEQ ID NO: 24)CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCGGAAATATTCTGGTATGATTATAGTGAAGCCACAGATGTATAATCATACCAGAATATTTCCTTGCC TACTGCCTCGGAATTCORC2mi1903:  (SEQ ID NO: 25)CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGAGGAACTGATGGAGTAGAGTATTAGTGAAGCCACAGATGTAATACTCTACTCCATCAGTTCCCTGCC TACTGCCTCGGAATTCORC2mi1981:  (SEQ ID NO: 26)CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGAGAGGCTTGAAGCTTTCCTTTATAGTGAAGCCACAGATGTATAAAGGAAAGCTTCAAGCCTCCTGCC TACTGCCTCGGAATTCEBNA1mi1666:  (SEQ ID NO: 27)CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCGTCCATTGTCTGTTATTTCATTAGTGAAGCCACAGATGTAATGAAATAACAGACAATGGACTTGCC TACTGCCTCGGAATTCLucifmi203:  (SEQ ID NO: 28)CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCCGATATGGGCTGAATACAAATTAGTGAAGCCACAGATGTAATTTGTATTCAGCCCATATCGTTGCC TACTGCCTCGGAATTC

5.1.3 Preparation of VSVG-Pseudotyped Amphotrophic Retrovirus

One million four hundred thousand Phoenix-amphotrophic packaging cells(cell line kindly provided by Gary Nolan) were seeded per well of 6-wellplates in 2 mL DMEM+10% fetal bovine serum (FBS). After allowing ˜24hours for cells to attach to wells, each culture was co-transfectedusing lipofectamine 2000 (Invitrogen) with pHelper plasmid, pVSVGplasmid, and a pLM-puro plasmid encoding a particular shRNA. The pHelperand pVSVG plasmids were added to improve infection efficiency of HCT116cultures. At ˜24 hours post-transfection, the transfection media on eachculture was aspirated and replaced with DMEM media+10% FBS. At ˜36 hourspost-transfection, media containing virus was collected from eachpackaging cell culture and filtered through a 0.45 micron filter using a3 mL syringe. Two milliliters DMEM+10% FBS was then added back to eachculture of packaging cells. At ˜42-48 hours post-transfection, mediacontaining virus was again collected from each packaging cell cultureand filtered through a 0.45 micron filter using a 3 mL syringe.Packaging cultures were then bleached and discarded according tostandard BL2+ safety protocol for disposal of infectious waste.

5.1.4 Infection and Selection of HCT116 Cultures

About 24 hours prior to infection, 150,000 HCT116 cells were seeded perwell of a 24-well plate in 0.4 mL DMEM+10% FBS. At the time of the firstround of infection, culture media was aspirated from each HCT116 cultureand 0.5 mL media containing virus, obtained from ˜36-hourpost-transfection packaging cell culture as described above, was addedto each HCT116 culture. Each HCT116 culture was infected with virusencoding a particular shRNA. The cultures were returned to theincubator, and ˜8-12 hours later, the media containing virus wasaspirated from each culture and replaced with media containing virusencoding the same shRNA, which was obtained from the ˜42-48-hourspost-transfection packaging cell cultures. Thus each HCT116 cultureunderwent two rounds of infection with virus encoding a particularshRNA.

About 24 hours after the second round of infection of the HCT116cultures, cells from each culture were suspended and seeded to 6-wellplates in 2 mL DMEM+10% FBS+1.5 μg/mL puromycin per well. Following twodays of growth, cells in each culture were suspended in 3 mLDMEM+FBS+1.5 μg/mL puromycin. Two milliliters of each suspension wereseeded to 6-well plates. Following two more days of growth (day 5post-infection), cells in each culture were suspended in complete DMEMand cell numbers per culture were counted using a hemacytometer.

5.1.5 Normalization of Results for Experimental RNAi Cultures

Results from the counting described above for 255 HCT116 culturestransduced with different shRNAs were generated from multipleexperiments. In each experiment, the impact of 36-48 different shRNAs onHCT116 proliferation was tested.

In each experiment, HCT116 cells were also transduced with hairpinsagainst EBNA1 (EBNA1mi1666) and Ff-luciferase (lucifmi203). 2-3 HCT116cultures were infected with virus encoding EBNA1mi1666; another 2-3HCT116 cultures were infected with virus encoding Lucifmi203. NeitherEBNA1 nor Ff-luciferase is encoded in the genome of HCT116 cells;therefore shRNAs targeting these genes should not impair HCT116proliferation. A BLAST search for sequences in the human genome thatmatch the sequences of these shRNAs was performed to verify that theseshRNAs would not target endogenous transcripts for degradation. Similarto cultures transduced with other shRNAs, on day 5 post-infection,HCT116 cells in cultures transduced with either EBNA1mi1666 orLucifmi203 were suspended and counted using a hemacytometer. HCT116cultures transduced with either EBNA1mi1666 or Lucifmi203 containedsimilar numbers of cells by day 5 post-infection, supporting theconclusion that neither shRNA impaired HCT116 proliferation.

The average cell number in HCT116 cultures transduced with EBNA1mi1666and Lucifmi203 was calculated for each experiment and cell numbers incultures transduced with the other shRNAs in the given experiment werenormalized to this value. These results are presented in FIG. 1. Sinceall results are normalized to the average cell number observed incultures transduced with EBNA1mi1666 and Lucifmi203, results for thesenegative control cultures have a value of 1.0 and are not presented onthe plot.

5.2 Example 2 Cytotoxicity of shRNAs in Human Cancer Cells

The assay described in Example 1 was used to identify shRNAs against DNAreplication proteins that had the greatest anti-proliferative effect onHCT116 cell cultures. FIG. 2 shows those hairpins with greater thantwo-fold inhibition of proliferation. shRNAs targeting RRM1, RPA1, RPA3,and PES1 were effective. See FIG. 2 and FIG. 3. FIG. 4 shows a Westernblot analysis of expression of RPA subunits in HCT116 culturestransformed with RNAi molecules against RPA3 and RPA1. RPA3 expressionis abolished by two shRNAs targeting RPA3.

TABLE 1 Cell numbers in experimental shRNA transduced cultures at 5 dayspost-infection (normalized to average of cells counted in negativecontrol shRNA transduced cultures) Cell # in culture/ average cellnumber in Gene shRNA ID negative control cultures RRM1 V2HS_938850.06872852 RPA1 V2HS_32160 0.10996564 RPA3 V2HS_32105 0.16494845 PES1V2HS_254080 0.17721519 PES1 V2HS_196673 0.27848101 RPA3 V2HS_321010.3161512 PCNA V2HS_152708 0.35738832 CDCA5 V2HS_70809 0.36426513 PES1V2HS_253534 0.39240506 DDX5 V2HS_24065 0.40506329 C8ORF1 V2HS_151190.41772152 dkfzp434b168 V2HS_96517 0.42268041 C8ORF1 V2HS_151180.44303797 MCM5 V2HS_84917 0.44620253 FLJ10154 V2HS_135540 0.46017699SLD5 V2HS_138608 0.46518987 C9orf76 V2HS_176960 0.49484536 dkfzp313a243V2HS_122245 0.50515464 ELLS1 V2HS_37210 0.50515464 FLJ20530 V2HS_1742040.50737463 FLJ20516 V2HS_220099 0.51327434 FLJ25416 V2HS_297990.53097345 TYMS V2HS_229638 0.53608247 dkfzp313a243 V2HS_1112310.53608247 KIAA1614 V2HS_59045 0.55457227 SLD5 V2HS_236484 0.56012658TYMS V2HS_94107 0.56357388 ZNF183 V2HS_95049 0.56701031 PCNA V2HS_1527100.57731959 c14orf130 V2HS_203406 0.57731959 RHOBTB3 V2HS_959430.57911392 RAMP V2HS_225424 0.59106529 USP53 V2HS_19252 0.59810127 CDC45V2HS_172575 0.604811 ELLS1 V2HS_37208 0.604811 C8ORF1 V2HS_151170.60759494 MCM5 V2HS_84918 0.60759494 MCM10 V2HS_175971 0.60759494 PSF1V2HS_34186 0.60759494 ORC5 V2HS_152497 0.61708861 RRM1 V2HS_938800.6185567 FLJ20516 V2HS_222044 0.63716814 MCM3 V2HS_151629 0.64556962RAMP V2HS_115868 0.64604811 CDCA7 V2HS_57684 0.64604811 MGC40214V2HS_21213 0.64948454 PSF1 V2HS_34187 0.65506329 Luzp5 V2HS_1736690.66475645 KIAA1614 V2HS_248717 0.67256637 FLJ10154 V2HS_2376860.68436578 CDCA5 V2HS_70812 0.68472622 dkfzp434b168 V2HS_965200.68728522 FLJ25416 V2HS_29802 0.69616519 MYCBP2 V2HS_254277 0.69620253C6orf72 V2HS_205077 0.6991404 MGC2610 V2HS_111372 0.70103093 TTC14V2HS_85344 0.7054755 ORC2 Orc2mi1903 0.71150436 MCM5 V2HS_849200.71202532 MCM10 V2HS_175970 0.71202532 FLJ20105 V2HS_155113 0.72206304RHOBTB3 V2HS_95941 0.73101266 MCM3 V2HS_151632 0.73101266 MCM10V2HS_221770 0.73101266 MGC2610 V2HS_111373 0.73195876 MYCBP2 V2HS_2004480.73417722 DDX5 V2HS_24063 0.73417722 CANP V2HS_285907 0.73775216MGC2610 V2HS_246266 0.74226804 Luzp5 V2HS_173665 0.74269341 C6orf72V2HS_205290 0.74498567 WDHD1 V2HS_199940 0.74498567 RHOBTB3 V2HS_959440.75 ORC3 V2HS_48977 0.75398223 KIAA1598 V2HS_175062 0.75516224 TIMELESSV2HS_47526 0.75601375 C9orf76 V2HS_176963 0.75601375 USP53 V2HS_192540.75949367 KDELC1 V2HS_98989 0.76103152 KIAA1529 V2HS_19447 0.76696165c14orf130 V2HS_203155 0.76975945 SLD5 V2HS_138603 0.77848101 Luzp5V2HS_277726 0.77936963 C6orf72 V2HS_250508 0.79083095 RBBP4 V2HS_570910.79725086 KIAA0701 V2HS_50916 0.79725086 KIAA0738 V2HS_2606270.79725086 CDC6 V2HS_112878 0.79725086 C1orf63 V2HS_39946 0.79725086USP53 V2HS_19251 0.79746835 FLJ10154 V2HS_135542 0.80235988 NT5C2L1V2HS_38659 0.81152738 kiaa0008 V2HS_95173 0.81375358 DC13 V2HS_509780.81375358 KIAA1529 V2HS_19475 0.81415929 KIAA1614 V2HS_2493840.81415929 FLJ20530 V2HS_174203 0.81415929 FLJ11806 V2HS_1580090.82474227 c14orf130 V2HS_202843 0.82474227 C1orf63 V2HS_399430.82474227 dkfzp434b168 V2HS_96521 0.82474227 KDELC1 V2HS_2316650.8252149 KDELC1 V2HS_98994 0.8252149 FLJ13231 V2HS_82233 0.8259587FLJ13231 V2HS_256796 0.8259587 CANP V2HS_285266 0.82997118 dkfzp313a243V2HS_122242 0.83505155 FLJ20530 V2HS_174199 0.83775811 FLJ11127V2HS_176291 0.83775811 KIAA1586 V2HS_102262 0.83775811 FLJ20105V2HS_155116 0.84813754 ORC2 Orc2mi1981 0.84955745 ORC4 V2HS_1524880.84955745 KIAA1598 V2HS_222689 0.84955752 RBBP4 V2HS_247612 0.85223368MCM3 V2HS_262054 0.85443038 SLD5 V2HS_138605 0.85443038 FBXL20V2HS_263207 0.8556701 FLJ20530 V2HS_223307 0.86135693 C9orf76V2HS_276972 0.86597938 dkfzp313a243 V2HS_225310 0.86597938 ZNF183V2HS_95052 0.86597938 ZNF183 V2HS_95051 0.86597938 ORC1 mi550 0.87187265MYCBP2 V2HS_251405 0.87341772 MGC2610 V2HS_111370 0.87972509 C1orf63V2HS_39941 0.87972509 ABHD10 V2HS_175389 0.88760807 ABHD10 V2HS_1753910.88760807 DC13 V2HS_50979 0.89398281 WDHD1 V2HS_196383 0.89398281FIGNL1 V2HS_201768 0.89398281 KIAA1529 V2HS_19426 0.89675516 FLJ11127V2HS_277435 0.89675516 XM_066946 V2HS_23889 0.90855457 ORC3 V2HS_489720.91327426 KDELC1 V2HS_98993 0.91690544 kiaa0008 V2HS_232596 0.91690544RBBP4 V2HS_57090 0.91752577 KIAA1529 V2HS_19402 0.92035398 XM_066946V2HS_23890 0.92035398 NUP43 V2HS_157038 0.9221902 FLJ20105 V2HS_1551150.92836676 PARP16 V2HS_174138 0.92836676 FLJ25416 V2HS_29798 0.93215339KIAA1586 V2HS_102265 0.93215339 ABHD10 V2HS_175390 0.93371758 FLJ13231V2HS_82234 0.9439528 ELLS1 V2HS_37211 0.94845361 ORC5 V2HS_1524940.94936709 UHRF1 V2HS_249046 0.9512894 PCNA V2HS_261970 0.96219931 CDC45V2HS_172574 0.96219931 CDCA7 V2HS_57686 0.96219931 FIGNL1 V2HS_2019150.96275072 PARP16 V2HS_174136 0.96275072 PARP16 V2HS_174137 0.96275072ORC3 V2HS_246135 0.9663716 kiaa0008 V2HS_95176 0.97421203 C10orf70V2HS_221723 0.97421203 FIGNL1 V2HS_201952 0.97421203 KIAA1598V2HS_223530 0.97935103 FLJ25416 V2HS_29797 0.97935103 NT5C2L1 V2HS_386580.97982709 UHRF1 V2HS_70516 0.98567335 ORC4 V2HS_152489 0.98761053 ORC6V2HS_199171 0.98761053 CDC6 V2HS_112875 0.98969072 CDCA7 V2HS_576820.98969072 FLJ35740 V2HS_47350 0.99115044 FLJ11127 V2HS_2207250.99115044 DDX11 V2HS_24008 0.99713467 NME1 V2HS_76045 1 COMMD7V2HS_88587 1.00288184 XM_066946 V2HS_23885 1.00294985 MGC2610 V2HS_493521.00343643 NME1 V2HS_76050 1.01030928 FBXO30 V2HS_202750 1.01440922NT5C2L1 V2HS_159134 1.01440922 KIAA1529 V2HS_19427 1.01474926 FLJ20516V2HS_174169 1.01474926 FLJ13231 V2HS_82235 1.01474926 MGC40214V2HS_21211 1.01718213 MGC2610 V2HS_111371 1.01718213 C1orf63 V2HS_399421.01718213 WDHD1 V2HS_6044 1.02005731 WDHD1 V2HS_198756 1.02005731COMMD7 V2HS_259262 1.0259366 PCNA V2HS_152712 1.03092784 CDC45V2HS_172579 1.03092784 Luzp5 V2HS_173668 1.04297994 FLJ12973 V2HS_1767831.04467354 CANP V2HS_285579 1.04899135 ATAD2 V2HS_54362 1.04899135NT5C2L1 V2HS_159132 1.04899135 KIAA0101 V2HS_73018 1.05063291 HBOIV2HS_197280 1.05063291 CROP V2HS_65519 1.05444126 DC13 V2HS_509821.05444126 C10orf70 V2HS_276497 1.05444126 FLJ12973 V2HS_1767821.05841924 MGC40214 V2HS_21212 1.05841924 dkfzp313a243 V2HS_1112351.05841924 RAMP V2HS_115869 1.05841924 KIAA1529 V2HS_19423 1.0619469HBOI V2HS_48974 1.06329114 CROP V2HS_65523 1.06361032 DDX11 V2HS_240051.06590258 LMO4 V2HS_84510 1.07204611 c14orf130 V2HS_255281 1.07216495DDX11 V2HS_242050 1.0773639 FBXO30 V2HS_202507 1.08357349 NUP43V2HS_157040 1.08357349 CDC6 V2HS_112873 1.08591065 COMMD7 V2HS_2595961.09510086 FBXL20 V2HS_195454 1.10309278 ORC6 V2HS_12700 1.10442468ATAD2 V2HS_54361 1.11815562 LMO4 V2HS_84513 1.11815562 FLJ35740V2HS_206342 1.12094395 ORC4 V2HS_235462 1.12566362 FLJ12973 V2HS_1767791.12714777 LMO4 V2HS_256362 1.129683 ORC6 V2HS_198092 1.13628309 ACDV2HS_116355 1.14121037 NUP43 V2HS_157037 1.14121037 FIGNL1 V2HS_2022761.14613181 FBXO30 V2HS_262021 1.15273775 FBXO30 V2HS_203571 1.15273775DDX17 V2HS_203066 1.15273775 FBXL20 V2HS_262023 1.15463918 CDCA5V2HS_70776 1.16426513 DDX17 V2HS_238845 1.16426513 dkfzp313a243V2HS_111234 1.16838488 C10orf70 V2HS_221836 1.16905444 ACD V2HS_1163561.17579251 FLJ20516 V2HS_174170 1.179941 ACD V2HS_116352 1.18040346KIAA0701 V2HS_50917 1.20962199 KIAA0701 V2HS_50914 1.20962199 KIAA0738V2HS_73695 1.20962199 TTC14 V2HS_85339 1.21037464 HBOI V2HS_1976761.21518987 ORC6 V2HS_199450 1.22123883 DDX17 V2HS_203274 1.22190202FBXO30 V2HS_202575 1.23342939 dkfzp313a243 V2HS_111232 1.2371134KIAA0101 V2HS_73015 1.24050633 NT5C2L1 V2HS_38657 1.25648415 FBXO30V2HS_262218 1.26801153 TTC14 V2HS_85343 1.26801153 NT5C2L1 V2HS_386561.2795389 WDHD1 V2HS_196266 1.28366762 FLJ11806 V2HS_158008 1.29209622ATAD2 V2HS_54364 1.33717579

5.3 Example 3 Cytotoxicity of siRNAs in Human Cancer Cells

siRNAs derived from the shRNAs of Example 2 were all highly cytotoxic toHCT116 cells. The siRNA sequences and shRNAs from which they werederived are shown below

RPA3 (SEQ ID NO: 1) RPA3si1417: CAUCUUAUGUCCAGUUUAA? (v2HS_32101)(SEQ ID NO: 2) RPA3si1403: CACCAUCUUGUGUACAUCU? (v2HS_32105) RRM1(SEQ ID NO: 3) RRM1si1820: CAGAUCUUUGAAACUAUUU? (v2HS_93885) CDC45(SEQ ID NO: 4) CDC45si384: CAGUCAAUGUCGUCAAUGUAU? (v2HS_172575) 

The numbers refer to the location in the cDNA sequences for therespectivegenes that these sequences initiate. In parenthesis are theCodex and Open Biosystems catalog numbers for the shRNAs from whichthese siRNAs were derived.

Cytotoxicity was determined by the reduction in culture confluency overtime after transfection of the siRNA relative to negative control RNAicultures. Interestingly, in the shRNA context V2HS_(—)32105 was morecytotoxic than V2HS_(—)32101. However in the siRNA context the oppositewas the case, where RPA3si1417 was more cytotoxic than RPA3si1403.

Using a slightly different assay, we observed about a 5-fold reductionin the ability of HCT116 cells transduced with V2HS_(—)172575 (targetingCDC45) to form colonies compared to cells transduced with the negativecontrol shRNAs, which was interpreted as a significant reduction inHCT116 proliferation. After selecting for puromycin resistant HCT116cells transduced with the shRNA, cultures were split and seeded 5,000cells into a well of a 12-well plate in media containing puromycin.Cultures were allowed to grow for 8 days, at which time puromycinresistant colonies were apparent. Wells were then stained with crystalviolet allowing visualization of any effect of the shRNA on colonyformation in comparison to control cell transduced with shRNAs againsteither Ff-luciferase or EBNA1. Crystal violet was destained off thecolonies and the amount of stain taken up by the well was measured,allowing quantitatation of the amount of colony formation in differentwells. Western blots of cells transduced with CDC45 shRNAs showed acorrelation between cytotoxicity and knockdown of CDC45 expression, withV2HS_(—)172575 being the most cytotoxic.

5.4 Example 4 RNAi Against DNA Replication Genes in Mouse Cells

An assay similar to that described in Example 1 was used to test theeffect of RNAi molecules against genes encoding DNA replication proteinsin mouse cancer cells (Hepa1.6). Similar results in mouse cells wereobserved as compared to human cells (FIG. 8 and FIG. 9). For example,mouse tumors regress upon induction of RNAi against Rpa3 (FIG. 17).

Results also showed that inducible RNAi targeted against Rpa3 causesregression in a genetically defined mosaic mouse model of human AML(FIG. 18).

Recipient cells expressing a shRNA against a target gene (for example,RPA3) are sorted based on a selectable marker whose expressionsubstantially matches the expression of the RNAi molecule (FIG. 5).Depletion for the target gene RPA3 is observed (for example, see FIGS.6A-C and FIG. 7).

5.5 Example 5 Additional Screens

A large-scale screen of cancer-related genes for modifiers ofproliferation in a mouse model of liver carcinoma identified a smallnumber of anti-proliferative shRNAs targeting ribosomal proteins (Rpl15,Rps4x) (FIGS. 11A-B). This result echoes the identification of ribosomalbiogenesis gene pescadillo in the human replication gene screen (FIG. 2and FIG. 3).

RNAi screening as described herein followed by in vivo validation, asdescribed below, to confirm the potentcy of therapeutic effect of aparticular RNAi molecule in vivo can be used to identify drug targetsfor cancer therapy. For example, a screen of Mixed Lineage Leukemia(MLL) pathway genes for modifiers of proliferation in an MLL-drivenmouse leukemia showed that shRNAs targeting c-Myb were stronglyantiproliferative (FIGS. 10A-F). Inducible RNAi against Myb causesregression of this aggressive, chemo-resistant leukemia in vivo (FIG.19).

5.6 Example 6 AML Mouse Model

We developed AML mouse models to reflect common genetic themes in humanAML. We used mice harboring leukemias that accurately reflect thegenetics and pathology of human AML as tractable models to study theimpact of cancer heterogeneity on therapy response and to explore themolecular basis for variable leukemia behavior. We see that leukemiasexpressing the AML1/ETO9a oncoprotein (mimicking translocation (8;21) inhumans) respond well to conventional chemotherapy, whereas thoseexpressing MLL/ENL (recapitulating translocation (11;19) in humans) showa particularly dismal response. Remarkably, the response patterns ofthese two leukemia subtypes accurately mimic what is observed in humanpatients with the same genetic lesions.

To rapidly generate mice harboring leukemias with various geneticalterations we applied a “mosaic” approach involving retroviraltransduction of oncogenes into hematopoietic stem- and progenitor cells,followed by re-transplantation of the genetically-altered cells intosyngeneic recipients (FIG. 30D). This strategy enables multipleoncogenes and reporter elements to be introduced in a one-stepprocedure, thereby facilitating disease monitoring and reducing theanimal husbandry associated with germline transgenic mice. To modeltranslocation (8;21) we choose to express the AML1/ETO9a splice variantthat is expressed in the majority of t(8;21) patients and has increasedleukemogenic potential in mice (Yan et al. 2006; Lafiura et al. 2008).To model MLL translocations, we used the MLL/ENL variant which is amongthe most common and associated with a particularly poor prognosis(Schoch et al. 2003). Both fusion genes were co-expressed with enhancedgreen fluorescent protein (EGFP) in bicistronic constructs (FIG. 30C).Oncogenic Nras^(G12D) was co-expressed with luciferase to enable imagingof the resulting leukemias by bioluminescence. Of note, Nras^(G12D) wascloned downstream of an internal ribosomal entry site (IRES), whichreduces expression of the ectopic Nras gene to near physiological levels(Parikh et al. 2006).

Each oncogene was introduced alone or in combination into fetal livercells (FLCs) derived from E13.5-15.5 embryos, and the infectedpopulations—which are highly enriched for hematopoietic stem andprogenitor cells (Morrison et al. 1995)—were used to reconstitute thehematopoietic compartment of lethally-irradiated syngeneic recipientmice (FIG. 30D). Recipient animals were then monitored for disease onsetby analysis of peripheral blood and bioluminescence for the transducedluciferase reporter. As expected, mice receiving FLCs expressingAML1/ETO9a or MLL/ENL alone eventually developed GFP-positiveleukocytosis consistent with peripheral leukemia. The survival of theseanimals was similar to previous reports, with AML1/ETO9a inducingleukemia with a longer latency and reduced penetrance compared toMLL/ENL (170 vs. 48 days median survival, p<0.0001).

Retroviral Constructs

All retroviruses were constructed in the MSCV backbone (Clontech).MSCV-IRES-Luciferase was generated by replacing GFP in MSCV-IRES-GFPwith an NcoI/SalI fragment from pGL3-Basic (Promega) after excision ofthe 3′polyA-signal. Mouse Nras^(G12D) was amplified and mutated by PCRfrom IMAGE clone 6475312 (Invitrogen) and cloned downstream of theinternal ribosomal entry site (IRES); subsequently Luciferase was clonedupstream of the IRES. The AML1/ETO9a cDNA was generated by PCR fromfull-length human AML1/ETO-IRES-GFP and subcloned into MSCV-IRES-GFP.Human MLL/ENL was subcloned from MLL/ENL-PGK-Neo (generously provided byIrving L. Weissman) into MSCV-IRES-GFP and MSCV-IRES-Luciferase.

MSCV-5′LTR-rtTA3-IRES-MLL/AF9 was constructed with the MSCV backbone(Clontech) with a standard EMCV (Encephalomyocarditis virus)-IRES(internal ribosomal entry site) to allow for bicistronic transgeneexpression. A reverse tet-transactivator, rtTA3 (Das, et al., JBC,2004), was cloned directly after the packaging signal and is driven bythe retroviral MSCV-LTR promotor assuring high expression levels.MLL/AF9 is a fusion oncogene isolated from a patient carrying a t(9;11)translocation. It also is expressed from the LTR-transcript and usesIRES as a secondary translational start.MSCV-5′LTR-Luciferase-IRES-Nras(G12D) was also constructed the MSCVbackbone (Clontech) with a standard EMCV (Encephalomyocarditisvirus)-IRES (internal ribosomal entry site) to allow for bicistronictransgene expression and comprises a Luciferase insert cloned frompGL-Prom (Promega) and mouse oncogenic Nras(G12D), which was cloned andmutated from a full-length cDNA clone (Invitrogen).

Although Nras^(G12D) was not oncogenic on its own, it cooperated withboth AML1/ETO9a and MLL/ENL to accelerate leukemia onset and reduceoverall survival (FIG. 30E), 170 to 84 days for AML1/ETO9a, p=0.0026; 48to 24 days for MLL/ENL, p<0.0001). Of note, leukemias arising from FLCstransduced with both retroviral vectors were invariably positive forboth luciferase (FIG. 30F) and GFP, suggesting a strong selectiveadvantage for cells harboring a fusion protein together with Nras.Indeed, the resulting leukemias expressed the expected transgenes (FIG.30G), and those harboring activated Nras displayed hyperactive MitogenActivated Protein Kinase (MAPK) signaling as assessed by flow cytometryfor phospho-Erk (a downstream Ras effector in the MAPK cascade) (FIG.30H). Thus, lesions that occur together in human AML cooperate torapidly generate leukemia in mice.

Fetal Liver Cell Isolation, Retroviral Transduction and Transplantation

E13.5-15.5 fetal liver cells from wild-type C57BL/6 mice were isolated,cultured and retrovirally transduced as previously described (Schmitt etal. 2002). Retroviral cotransduction was carried out by mixing viralsupernatants from independent transfections of Phoenix packaging cellsin a 1:1 ratio. Retroviral transduction of GFP expressing constructs wasassessed 24 h after the last infection by flow cytometry (GuavaEasyCyte, Guava Technologies); typically demonstrating transduction ofabout 20% of fetal liver cells. Transduction of luciferase expressingconstructs was confirmed qualitatively (IVIS100, Caliper LifeSciences).About 1×10⁶ cells were transplanted by tail vein injection of 6-8 weekold lethally irradiated C57BL/6 recipient mice (9.0 Gy as single doseadministered 24 h prior to transplantation). The Cold Spring HarborAnimal Care and Use Committee approved all mouse experiments included inthis work.

Monitoring and Characterization of Primary Leukemias

Bioluminescent imaging was performed using an IVIS100 imaging system(Caliper LifeSciences). Mice were injected intraperitoneally with 150mg/kg D-Luciferin (Caliper LifeSciences), anesthetized with isofluraneand imaged for 1 minute after a 5 minute incubation following injection.Primary FLC recipient mice were sacrificed at terminal disease stage(whole body signal in bioluminescent imaging, severe leukocytosis inperipheral blood smears, moribund appearance) by CO₂ euthanasia.Leukemia cells were harvested from bone marrow (by flushing tibias andfemurs with DMEM) and spleen (by gently meshing enlarged spleens betweentwo glass slides), filtered through a nylon screen (35 μm) to obtainsingle-cell suspensions and cryopreserved in fetal bovine serum (FBS)containing 10% DMSO. Peripheral blood smears and bone marrow cytospinswere stained with Wright-Giemsa (Sigma); tissue specimens were fixed in10% formalin and stained with hematoxylin and eosin (Sigma) usingstandard protocols.

For immunophenotyping, single-cell suspensions of bone marrow and spleenwere incubated in ACK red cell lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃,0.1 mM EDTA) for 5 minutes, resuspended in FACS buffer (PBS, 5% FBS,0.1% NaN₃) and preincubated with anti-CD16/CD32 (F_(c)-block, 1:1000, BDPharmingen). Aliquots of ˜0.5×10⁶ cells were stained for 20 minutes onice with phycoerythrin-CyS-conjugated monoclonal antibodies specific forMac-1, Gr-1, F4/80, CD45, CD3, CD19, B220, TER119, CD117, Sca-1 and aphycoerythrin-conjugated Ly-6C antibody (all 1:100, Biolegend). Datawere collected on a LSR-II flow cytometer (BD Biosciences) and analyzedusing FACSDiva (BD Biosciences) and FlowJo (Treestar) software.Statistical evaluation of overall survival was based on the log-rank(Mantel-Cox) test for comparison of the Kaplan-Meier event-time format.

5.7 Example 7 Murine Leukemias Display Pathological Features of HumanAML

In human patients, AML is characterized by the appearance of malignantmyeloid progenitors, which rapidly accumulate in bone marrow andaggressively infiltrate extramedullary tissues. Mice harboring leukemiasco-expressing either AML1/ETO9a or MLL/ENL together with oncogenic Nrasdisplayed progressive anemia and leukocytosis (See FIG. 31A), withextensive hepatosplenomegaly. Upon reaching a terminal stage,GFP-positive cells dominated their peripheral blood, bone marrow, spleenand liver (FIGS. 31C-D) Immunophenotyping, cytologic and histologicanalyses revealed that AML1/ETO9a+Nras leukemias predominantly containimmature blasts (Mac1⁻, Gr1⁻, Ly-6C⁻, F4/80^(−/lo), CD45^(lo), CD3⁻,CD19⁻, B220⁻, TER119⁻, CD117⁺, Sca-1⁻; FIGS. 31B-C), which parallelsobservations in other models of core-factor binding AML (Yan et al.2004; Kuo et al. 2006). Although such purely immature subtypes are seenin t(8;21)-positive AML, most human cases show a significantgranulocytic maturation besides immature myeloblasts, which was absentin murine AML1/ETO9a+Nras AML.

In contrast, MLL/ENL+Nras leukemias predominantly consisted of moredifferentiated myeloid cells (Mac1⁺, Gr1^(+/lo), Ly-6C⁺, F4/80^(+/lo),CD45⁺, CD3⁻, CD19⁻, B220⁻, TER119⁻, CD117^(lo/−), Sca-1⁻, FIGS. 31B-C)and involve the monocytic lineage—a typical feature of human11q23-rearranged AML. Similar to previous MLL fusion-based mouse models(Cozzio et al. 2003), the morphology resembles myelomonocytic andmonocytic subtypes, which are seen in ˜42% of human cases of11q23-rearranged AML (Schoch et al. 2003). Despite its potent effects onleukemia onset, Nras did not appear to influence leukemia morphology,since leukemias induced by AML1/ETO9a or MLL/ENL alone were similar tothose co-expressing Nras. In summary, mosaic mouse models based oncooperation of Nras with either fusion protein recapitulate commongenetic and pathologic features of human AML.

5.8 Example 8 Establishment of Murine Chemotherapy Regimen that MimicsClinical AML Induction Therapy

To study factors that influence the outcome of conventional AML therapyin mice, we established a simple chemotherapy regimen involvingintraperitoneal injection of cytarabine (100 mg/kg over 5 days) anddoxorubicin (3 mg/kg over 3 days; FIG. 36A), which mimics the standardinduction therapy used to treat AML in human patients. Of note, in ourstudies, doxorubicin was used over the more commonly used anthracyclinedaunorubicin, which showed high toxicity in mice if appliedintraperitoneally. Efficacy and safety of this regimen were initiallyevaluated in a previously described model based on co-transduction ofMLL/ENL and FLT3-ITD (Ono et al. 2005). In order to rapidly generatehomogenous treatment cohorts we harvested bone marrow and spleen fromterminally ill primary FLC recipient mice and transplanted 1 millionleukemia cells into 20-30 secondary recipients. In initial studies, wenoted that this combination therapy was not tolerated in mice whentreatment was initiated based on a high leukemia burden in theperipheral blood; although the peripheral disease disappeared within twodays, animals died shortly thereafter with peripheral leukopenia.

To better monitor leukemia progression and treat animals at an earlierstage of disease, we took advantage of the luciferase reporter, which welinked to MLL/ENL in a bicistronic retrovirus. Bioluminescent imagingallowed detection of MLL/ENL+FLT3-ITD leukemic cells in bone marrow,spleen and liver approximately 10 days after transplantation intosecondary recipients, which corresponded to approximately 30%infiltration in bone-marrow and spleen in pathologic analysis, while thedisease became apparent in peripheral bloodsmears about 5-7 days later(FIG. 36A). When mice were treated when disease was detected bybioluminescent imaging, most showed some response to therapy (FIG. 36B),while untreated controls progressed rapidly (FIG. 36C). Although mostresponses were only partial (median survival benefit of 7 days,p<0.0001; FIG. 36D), none of the animals appeared to succumb totreatment toxicity, but instead from advancing leukemia. These studiesdemonstrate the utility of bioluminescence monitoring for treatmentstudies and establish an effective and safe combined chemotherapyregimen that mirrors AML induction therapy.

Transplantation and In-Vivo Treatment Studies

For treatment studies each primary leukemia was transplanted into 20-306-8 weeks old sublethally irradiated recipient mice (4.5 Gy, 24 h priorto transplantation) by tail-vein injection of 1×10⁶ viableGFP+cells/recipient. Viability was assayed by propidium iodide (PI)staining; GFP+/PI− cells were counted using a Guava EasyCyte flowcytometer (Guava Technologies). Recipient mice were monitored bybioluminescent imaging every 4 days starting 10 days aftertransplantation. Treatment was initiated upon detection of clear signalsin pelvis, tail and both femurs and initial stage of hepatosplenicinfiltration, which correlated with 30-60% bone marrow infiltration asassessed by flow cytometry. Mice were treated for five consecutive daysevery 24 h with intraperitoneal (i.p.) injections of 100 mg/kgCytarabine (Bedford Laboratories); during the first three days 3 mg/kgDoxorubicin (Bedford Laboratories) was administered in a separate i.p.injection. Immediate response and long-term treatment effects weremonitored by weekly luciferase imaging starting the first day aftertreatment and by histopathological analysis of representative mice atvarious time points.

Gene Expression Analysis (Quantitative Real-Time PCR, Microarrays,Immunoblotting)

For drug-response studies, diseased mice were treated with a single doseof Cytarabine (100 mg/kg) and Doxorubicin (3 mg/kg) i.p. at various timepoints prior to harvest. Samples were obtained from enlarged spleenspredominantly containing leukemia cells; infiltration of >85% GFP+ cellswas confirmed by flow cytometry. Following ACK-lysis of single cellsuspensions, RNA was extracted using RNAeasy columns (Qiagen) and cDNAwas synthesized using TaqMan reverse transcriptase reaction (AppliedBosystems) according to the manufacturer's protocols. Quantitative PCRanalysis was performed on an iCycler mounted with an iQ5 multicolor realtime PCR detection system (Biorad). Primers used were specific forMLL/ENL (MLLf 5′-TCCCGCCTCAGCCACCTACTACAG (SEQ ID NO: 29), ENLr5′-CGTGGTGGGCTTCTTGCGCAGTT (SEQ ID NO: 30)), AML1/ETO9a (AML1f5′-CTACCGAGCCATGAAGAACC (SEQ ID NO: 31), ETOr 5′-AGAGGAAGGCCCATTGCTGAA(SEQ ID NO: 32)), Cdkn1a (p21f 5′-GTTCCGCACAGGAGCAAAGT (SEQ ID NO: 33),p21r 5′-ACGGCGCAACTGCTCAC (SEQ ID NO: 34)) and Mdm2 (Mdm2f5′-CTCACAGATTCCAGCTTCGGA (SEQ ID NO: 35), Mdm2r 5′-TGCGCTCCAACGGACTTTA(SEQ ID NO: 36)). Parallel reactions were done with primers specific forbeta-actin (actbf 5′-CTCTGTGTGGATCGGTGGCT (SEQ ID NO: 37), actbr5′-GCTGATCCACATCTGCTGGAAA (SEQ ID NO: 38)). Expression data werenormalized against actin controls. Microarray experiments were performedon Mouse Genome 430A 2.0 arrays (Affymetrix) according to manufacturer'sinstructions.

For Western blot analysis, ACK-treated samples were lysed in Laemmlibuffer, separated by SDS-PAGE and transferred to Immobilon PVDF membrane(Millipore). We used antibodies against p53 (IMX25, Leica Microsystems,1:1000), p21 (C-19, Santa Cruz Biotechnology, 1:500), Nras (F155, SantaCruz Biotechnology, 1:300), pan-Ras (Ras10, Upstate Biotechnology,1:1000) and Actin (AC-15, Abcam, 1:5000). Flow cytometric analysis ofphospho-Erk in bone marrow was performed using anti-pERK (#9101, CellSignaling, 1:100) as previously described (Van Meter et al. 2007). Toassess baseline phospho-Erk levels, cells were starved in the serum-freeharvesting medium at 37° C. for 30 min. and then fixed and analyzedwithout cytokine stimulation.

5.9 Example 9 AMLs Expressing AML1/ETO9a or MLL/ENL Display Differencesin Response to Conventional Chemotherapy

To determine whether defined AML genotypes display distinct treatmentsensitivities, we transplanted multiple primary AML1/ETO9a+Nras andMLL/ENL+Nras leukemias and treated secondary recipient mice uponbioluminescent disease detection in bone marrow and spleen. Leukemiascoexpressing AML1/ETO9a and Nras rapidly regressed under combinedchemotherapy leading to complete remissions. Thus, by 6 days afterinitiating treatment, luciferase signals disappeared completely, andhistological analyses and flow cytometry revealed that the bone marrowand spleen were leukemia-free (FIGS. 32A and 35B). In most instances,mice harboring these leukemias remained in remission for at least 30days (FIG. 32B), showing an overall survival benefit of 69 days(p<0.0001; FIG. 32C). Although 80% of the treated mice eventuallyrelapsed, often emanating from focal regions in the bone or spleen, therest remained leukemia free for even one year. Such behavior isreminiscent of the generally effective action of induction therapy inpatients.

In stark contrast, MLL/ENL+Nras induced AML persisted underchemotherapy. Bioluminescent imaging merely showed a deceleratedprogression, while histology and flow cytometry revealed refractoryleukemic infiltrates in the bone marrow (FIGS. 32A and 35B). After thisminor stagnation in disease acceleration, animals progressed rapidly anddied with only a minor survival benefit (5 days compared to untreatedcontrols, p<0.0001; FIG. 32C). Hence, the MLL/ENL fusion proteinproduced leukemias that showed a very poor therapeutic response.

Our studies find a remarkable similarity in the genotype-responsepattern of mouse AML models and human AML patients to standard inductionchemotherapy, implying that such systems can predict the behavior oftherapeutic agents in the clinic. Therefore, these and similar mousemodels provide relevant systems for further studies on drug action anddrug resistance and, by extrapolation, predictive test systems forcharacterizing potential new drug targets or testing new therapies fortheir efficacy at treating otherwise refractory malignancies.

5.10 Example 10 Screening and Evaluation of RNAi Targets

We applied tet-regulated in vivo RNAi to evaluate the role of candidatedrug target genes in established MLL-fusion leukemia. These results havecharacterized seven genes as essential for the survival of establishedMLL/AF9 induced AML, amongst them four MLL-associated genes (MLL, Men1,Meis1, Myb) that are dispensable in Mefs. These studies demonstrate thepower of tet-regulated RNAi to identify and evaluate genetic Achilles'heels in chemotherapy-resistant leukemia. To facilitate these studies weused the bioluminescent tet-on competent AML mouse model based oncooperation of an MLL-fusion oncogene (MLL/AF9) and oncogenic Nras,which reflects a common genetic association in human AML. Coexpressionof MLL/AF9 and Nras^(G12D) resulted in aggressive myelomonocyticleukemias (mean survival 21 days), which are refractory to combinedchemotherapy (see FIG. 12).

An especially advantageous feature of the tet-on competent AML mousemodel is in the use of tet-on competent leukemia cells, wherein saidtet-on competent leukemia cells carry a bicistronic nucleic acidconstruct comprising a promoter operably linked to a fusion geneassociated with chemotherapy-resistant leukemia, and a sequence encodinga reverse tet-transactivator protein, such that both coding sequencesare co-expressed from said promoter. Importantly, we determined that inusing inducible shRNA vectors for negative selection RNAi screening, asa critical prerequisite, it is essential to maintain stable, robustexpression of a tet transactivator protein in all transplanted leukemiccells. Otherwise, outgrowth of clones in which shRNA expression is nolonger inducible (for example, through selective pressure againstexpression of therapeutically active shRNAs) severely compromisessuccess of screening or evaluation of shRNA activity.

A critical feature of mosaic mouse model for negative selection RNAiscreening (see Examples 6-7) is the co-expression of a tettransactivator protein (e.g., rtTA3, a potent and non-toxic rtTAvariant) together with the oncogene responsible for maintaining theleukemic phenotype, (e.g. MLL/AF9), from one promotor (e.g. LTR) in abicistronic vector, ensuring that stable, robust rtTA expression andinducibility of shRNA expression is maintained in all transplantedcells, and independently of the identity of the particular shRNA beingexpressed.

Retroviral Constructs and Isolation of Tet-on AML Cells

SCV-5′LTR-rtTA3-IRES-MLL/AF9 was constructed with the MSCV backbone(Clontech) with a standard EMCV (Encephalomyocarditis virus)-IRES(internal ribosomal entry site) to allow for bicistronic transgeneexpression. A reverse tet-transactivator, rtTA3 (Das, et al., JBC,2004), was cloned directly after the packaging signal and is driven bythe retroviral MSCV-LTR promotor assuring high expression levels.MLL/AF9 is a fusion oncogene isolated from a patient carrying a t(9;11)translocation. It also is expressed from the LTR-transcript and usesIRES as a secondary translational start.MSCV-5′LTR-Luciferase-IRES-Nras(G12D) was also constructed in the MSCVbackbone (Clontech) with a standard EMCV (Encephalomyocarditisvirus)-IRES (internal ribosomal entry site) to allow for bicistronictransgene expression and comprises a Luciferase insert cloned frompGL-Prom (Promega) and mouse oncogenic Nras(G12D), which was cloned andmutated from a full-length cDNA clone (Invitrogen).

Tet-on leukemia (MLL/AF9+Nras AML) cells were obtained from a mousetransplanted with fetal liver cells derived from mouse embryos (e.g.,E13.5-15.5 embryos) and stably transformed with the bicistronic tet-onconstruct SCV-5′LTR-rtTA3-IRES-MLL/AF9. Fetal liver cell isolation,retroviral isolation, retroviral transduction and transplantation wereessentially carried out as above (see Examples 6-8).

To evaluate the potency of single-gene directed approaches in thesetherapy resistant AMLs, tumor cells were isolated from mice andtransduced with a series of tet-regulatable shRNAs targeting (i)essential genes involved in DNA replication [Rpa1, Rpa3, PCNA], (ii) MLLassociated genes [MLL, HoxA9, Meis1, Men1, Dot1L, Myb] and (iii) othercontrols [Luciferase, Braf]. Effects of doxycycline-induced shRNAexpression in vitro and in vivo were studied using a dual-colortet-shRNA vector that allows precise tracking of shRNA expressing cells.TRMPV was constructed in the pQCX1X self-inactivating retroviralbackbone (Clontech, Palo Alto, Calif.) by inserting the Tet-responsiveelement (TRE), dsRed, the miR30 context and a PGK-Venus expressioncassette using standard cloning techniques (FIG. 5). In someexperiments, variants of the vector an IRES-Neomycin (TRMPVIN) orIRES-Hygromycin (TRMPVIH) cassette were introduced downstream of Venusto facilitate drug selection. To construct TRMPVIR Neomycin in TRMPVINwas exchanged to rtTA3 (Das et al. 2004) and the TRE promoter wasreplaced by the TREtight variant (Clontech 2003). After selection, thetet-on cells (MLL/AF9+Nras AML) now carrying tet-inducible shRNAs weretransplanted into secondary recipient mice. (see FIGS. 13-14). Followingleukemia onset as evidenced through bioluminescent imaging mice weretreated with oral doxycycline to induce expression of these shRNAs inthe recipient mice.

In results of these experiments potent antileukemic effects wereobserved for multiple shRNAs targeting Rpa1, Rpa3, PCNA and certain MLLassociated genes (MLL itself, Meis1, Men1, Myb). For example, in someexperiments, mice were transplanted with AML cells carryingtet-inducible Rpa3 and Myb shRNAs. Following leukemia onset as evidencedthrough bioluminescent imaging, treatment of mice with oral doxycyclineinduced rapid and durable remissions upon induction of Rpa3 and MybshRNAs. Histological analysis of the bone marrow and liver showed thatshRNAs targeting Rpa3 or Myb ameliorated MLL/AF9-Nras leukemia (FIG. 15and FIG. 16). In contrast, mice transplanted with MLL/AF9+Nras AML cellsexpressing Braf control shRNAs suffered from continued progression ofleukemia under doxycycline treatment. In particular, these resultsdemonstrate that chemotherapy-resistant MLL/AF9-Nras leukemia can beameliorated by tet-inducible shRNAs targeting Rpa3 (and otherscomponents of the replication machinery) (FIG. 18), as well as Myb (seeFIG. 19). In parallel assays using tet-on competent Mefs we show thatthe antileukemic effects of shRNAs targeting MLL-associated genes arenot due to general cytotoxicity.

The shRNAs identified as having potent antileukemic effects are directlyuseful in various embodiments of the invention. Additionally, modifiedshRNA molecules and siRNA molecules directed against the targeted genesand derived from these shRNAs according to the disclosures herein (forexample, as exemplified in sections 4.3-4.5, Example 3) are useful inother embodiments of the invention, and in particular, in thetherapeutic use of such siRNA molecules to treat therapy resistantcancers. shRNA that exhibited particularly potent antileukemic effectsare shown below:

Bcl2.1132: (SEQ ID NO: 40)TGCTGTTGACAGTGAGCGCGACTGATATTAACAAAGCTTATAGTGAAGCCACAGATGTATAAGCTTTGTTAATATCAGTCTTGCCTACTGCCTCGGA Bcl2.1422:(SEQ ID NO: 41) TGCTGTTGACAGTGAGCGACCGGGAGAACAGGGTATGATATAGTGAAGCCACAGATGTATATCATACCCTGTTCTCCCGGCTGCCTACTGCCTCGGA Bcl2.2169:(SEQ ID NO: 42) TGCTGTTGACAGTGAGCGACAGTAGAAATTATATGCATTATAGTGAAGCCACAGATGTATAATGCATATAATTTCTACTGCTGCCTACTGCCTCGGA Mcl1.1334:(SEQ ID NO: 43) TGCTGTTGACAGTGAGCGAAAGAGTCACTGTCTGAATGAATAGTGAAGCCACAGATGTATTCATTCAGACAGTGACTCTTCTGCCTACTGCCTCGGA Mcl1.1792:(SEQ ID NO: 44) TGCTGTTGACAGTGAGCGAAACAGCCTCGATTTTTAAGAATAGTGAAGCCACAGATGTATTCTTAAAAATCGAGGCTGTTCTGCCTACTGCCTCGGA Mcl1.2018:(SEQ ID NO: 45) TGCTGTTGACAGTGAGCGCGGACTGGTTATAGATTTATAATAGTGAAGCCACAGATGTATTATAAATCTATAACCAGTCCATGCCTACTGCCTCGGA Meis1.1045:(SEQ ID NO: 46) TGCTGTTGACAGTGAGCGAAAGATGTAACAAGATCAGCAATAGTGAAGCCACAGATGTATTGCTGATCTTGTTACATCTTCTGCCTACTGCCTCGGA Meis1.2095:(SEQ ID NO: 47) TGCTGTTGACAGTGAGCGCTACGTTGTTTCTTATAGATTTTAGTGAAGCCACAGATGTAAAATCTATAAGAAACAACGTAATGCCTACTGCCTCGGA Meis1.2775:(SEQ ID NO: 48) TGCTGTTGACAGTGAGCGCTATGTAGACATTGTAAATAAATAGTGAAGCCACAGATGTATTTATTTACAATGTCTACATAATGCCTACTGCCTCGGA Men1.219:(SEQ ID NO: 49) TGCTGTTGACAGTGAGCGACGGAATTGTAAGGAACTAGAATAGTGAAGCCACAGATGTATTCTAGTTCCTTACAATTCCGGTGCCTACTGCCTCGGA Men1.2310:(SEQ ID NO: 50) TGCTGTTGACAGTGAGCGCCACCCTCATCCTCTAATTCAATAGTGAAGCCACAGATGTATTGAATTAGAGGATGAGGGTGATGCCTACTGCCTCGGA Men1.2707:(SEQ ID NO: 51) TGCTGTTGACAGTGAGCGACTGCCCGAATTTGGAAATCTTTAGTGAAGCCACAGATGTAAAGATTTCCAAATTCGGGCAGCTGCCTACTGCCTCGGA Myb.2572:(SEQ ID NO: 52) TGCTGTTGACAGTGAGCGCTCCATGTATCTCAGTCACTAATAGTGAAGCCACAGATGTATTAGTGACTGAGATACATGGAATGCCTACTGCCTCGGA Myb.2652:(SEQ ID NO: 53) TGCTGTTGACAGTGAGCGCCCCAAGTAATACTTAATGCAATAGTGAAGCCACAGATGTATTGCATTAAGTATTACTTGGGATGCCTACTGCCTCGGA Myb.670:(SEQ ID NO: 54) TGCTGTTGACAGTGAGCGACACAACCATTTGAATCCAGAATAGTGAAGCCACAGATGTATTCTGGATTCAAATGGTTGTGCTGCCTACTGCCTCGGA Pcna.1216:(SEQ ID NO: 55) TGCTGTTGACAGTGAGCGCAAGAAATATTGTTCAATTTAATAGTGAAGCCACAGATGTATTAAATTGAACAATATTTCTTATGCCTACTGCCTCGGA Pcna.538:(SEQ ID NO: 56) TGCTGTTGACAGTGAGCGCGGAGTACAGCTGTGTAATAAATAGTGAAGCCACAGATGTATTTATTACACAGCTGTACTCCTTGCCTACTGCCTCGGA Rpa1.1620:(SEQ ID NO: 57) TGCTGTTGACAGTGAGCGCCCGCATGATCTTATCGGCAAATAGTGAAGCCACAGATGTATTTGCCGATAAGATCATGCGGTTGCCTACTGCCTCGGA Rpa3.278:(SEQ ID NO: 58) TGCTGTTGACAGTGAGCGCAAGGAAGATACTAATCGCTTTTAGTGAAGCCACAGATGTAAAAGCGATTAGTATCTTCCTTATGCCTACTGCCTCGGA Rpa3.431:(SEQ ID NO: 59) TGCTGTTGACAGTGAGCGCAAGGAAGACTCCTGCAGTTTATAGTGAAGCCACAGATGTATAAACTGCAGGAGTCTTCCTTATGCCTACTGCCTCGGA Rpa3.457:(SEQ ID NO: 60) TGCTGTTGACAGTGAGCGCGCGACTCCTATAATTTCTAATTAGTGAAGCCACAGATGTAATTAGAAATTATAGGAGTCGCTTGCCTACTGCCTCGGA Rpa3.561:(SEQ ID NO: 61) TGCTGTTGACAGTGAGCGCAAAAGTGATACTTCAATATATTAGTGAAGCCACAGATGTAATATATTGAAGTATCACTTTTATGCCTACTGCCTCGGA

5.11 Example 11 An Epigenetics RNAi Screen for Novel Therapeutic Targetsin Chemotherapy-Resistant AML

As described above, in one non-limiting embodiment, shRNAs arecomplementary to a nucleotide sequence of a DNA replication protein.Non-limiting example of DNA replication proteins include replicationprotein A3 (RPA3), ribonucleotide reductase M1 (RRM1), cell divisioncycle 45 (CDC45) and pescadillo 1 (PES1). In another non-limitingembodiment, shRNAs are complementary to a nucleotide sequence ofepigenetic modifier genes. Here, we also applied tet-regulated in vivoRNAi in our MLL-fusion leukemia model to evaluate the role of epigeneticmodifier genes as candidate drug targets. To identify novel therapeutictargets for chemotherapy-resistant AML, we established a screeningmethod that utilizes a combination of in vitro and in vivo RNAitechnologies. We specifically investigated other enzymes that modifychromatin (epigenetic modifiers) as potential new drug targets in thisdisease. A custom library of 1,100 shRNAs was constructed that targetsall known epigenetic regulators that have been identified to date,amounting to 235 genes (FIG. 20). These shRNAs were cloned into the LMNmir30-embedded shRNA vector driven by constitutive retroviral LTRpromoter. A downstream Pgk promoter drives expression of Neomycinresistance cassette and eGFP separated by an internal ribosomal entrysite. The choice of neomycin resistance was to allow subsequent in vivotesting in the final stage of validation, as other drug resistancemarkers are rejected by an immune response in mice.

The primary screen was performed by systematically profiling each of the1100 individual shRNAs in the epigenetics library for its ability toconfer a proliferative disadvantage to MLL-AF9/Nras leukemia cell growthin vitro (FIG. 21). This was monitored by measuring the relative loss ofthe shRNA+/GFP+ population over 10 days following transduction of theleukemia cells. Each shRNA was scored by this method, which identified35 epigenetic regulators that were required for proliferation ofleukemia cell in vitro (based on a cutoff of 5-fold depletion and >2shRNAs identified per gene). To gain insight into whether therequirement for these genes was unique to leukemia cells or was ageneral feature of all non-transformed hematopoietic cells, we comparedthe ability of the identified shRNAs to inhibit growth of leukemiacells, non-transformed erythroid cells (G1E), non-transformed myeloidcells (32D), and non-transformed stem-like cells (EML). The result ofthis multi-parameter testing revealed 8 genes that leukemias require fortheir proliferation but is dispensable for growth of all otherhematopoietic cell lines tested (FIGS. 22-23). The findings are incontrast to the Rpa3 and Myc genes, which were required in all 4 celltypes for growth in vitro (FIG. 23). The identified genes are Eed,Suz12, Aof2, Smarca4, Smarcd1, Men1, Hdac3, and Whs111.

The sequence of the shRNAs from the library targeting these genes arelisted below as the oligo sequence. The knockdown efficiency of eachshRNA was tested and correlated with the relative inhibition of leukemiacell proliferation (data not shown). These shRNAs, identified as havingpotent and specific antileukemic effects are directly useful in variousembodiments of the invention. Additionally, modified shRNA molecules andsiRNA molecules directed against the targeted genes and derived fromthese shRNAs according to the disclosures herein (for example, asexemplified in sections 4.3-4.5, Example 3) are useful in otherembodiments of the invention, and in particular, in the therapeutic useof such siRNA molecules to treat therapy resistant cancers.

shRNA Sequences for AOF2:

Aof2.2435: (SEQ ID NO: 39)TGCTGTTGACAGTGAGCGCCTGGAAATGACTATGATTTAATAGTGAAGCCACAGATGTATTAAATCATAGTCATTTCCAGATGCCTACTGCCTCGGA Aof2.1153:(SEQ ID NO: 218) TGCTGTTGACAGTGAGCGAATGGCTGTCGTCAGCAAACAATAGTGAAGCCACAGATGTATTGTTTGCTGACGACAGCCATGTGCCTACTGCCTCGGA Aof2.1869:(SEQ ID NO: 219) TGCTGTTGACAGTGAGCGCAGGCTTGGACATTAAACTGAATAGTGAAGCCACAGATGTATTCAGTTTAATGTCCAAGCCTTTGCCTACTGCCTCGGA Aof2.2857:(SEQ ID NO: 220) TGCTGTTGACAGTGAGCGCTTGGAAGTACAGCTCCATAAATAGTGAAGCCACAGATGTATTTATGGAGCTGTACTTCCAAATGCCTACTGCCTCGGA Aof2.1956(SEQ ID NO: 221) TGCTGTTGACAGTGAGCGACACAAGTCAAACCTTTATTTATAGTGAAGCCACAGATGTATAAATAAAGGTTTGACTTGTGGTGCCTACTGCCTCGGA Aof2.2741(SEQ ID NO: 222) TGCTGTTGACAGTGAGCGACAAGCTCTTCTAGCAATACTATAGTGAAGCCACAGATGTATAGTATTGCTAGAAGAGCTTGCTGCCTACTGCCTCGGA

shRNA Sequences for EED:

Eed.949: (SEQ ID NO: 223)TGCTGTTGACAGTGAGCGACTGGATCTAGAGGCATTATAATAGTGAAGCCACAGATGTATTATAATGCCTCTAGATCCAGCTGCCTACTGCCTCGGA Eed.710:(SEQ ID NO: 224) TGCTGTTGACAGTGAGCGCCAGCCTCAAGGAAGATCATAATAGTGAAGCCACAGATGTATTATGATCTTCCTTGAGGCTGTTGCCTACTGCCTCGGA Eed.1397:(SEQ ID NO: 225) TGCTGTTGACAGTGAGCGCAGGCGATTTGATACTTTCCAATAGTGAAGCCACAGATGTATTGGAAAGTATCAAATCGCCTATGCCTACTGCCTCGGA Eed.1083:(SEQ ID NO: 226) TGCTGTTGACAGTGAGCGCAAAGATCATGCTTTACGGTTATAGTGAAGCCACAGATGTATAACCGTAAAGCATGATCTTTATGCCTACTGCCTCGGA Eed.1820:(SEQ ID NO: 227) TGCTGTTGACAGTGAGCGCTAGAAGTAATGTATCTTGCTATAGTGAAGCCACAGATGTATAGCAAGATACATTACTTCTATTGCCTACTGCCTCGGA Eed.1765:(SEQ ID NO: 228) TGCTGTTGACAGTGAGCGAATCGACTTCGATAAACTATTTTAGTGAAGCCACAGATGTAAAATAGTTTATCGAAGTCGATCTGCCTACTGCCTCGGA

shRNA Sequences for HDAC:

Hdac3.987: (SEQ ID NO: 229)TGCTGTTGACAGTGAGCGACCCGGTGTTGGACATATGAAATAGTGAAGCCACAGATGTATTTCATATGTCCAACACCGGGCTGCCTACTGCCTCGGA Hdac3.161:(SEQ ID NO: 230) TGCTGTTGACAGTGAGCGACTGGCATTGACTCATAGCCTATAGTGAAGCCACAGATGTATAGGCTATGAGTCAATGCCAGGTGCCTACTGCCTCGGA Hdac3.854:(SEQ ID NO: 231) TGCTGTTGACAGTGAGCGATCCCTGGGCTGTGATCGATTATAGTGAAGCCACAGATGTATAATCGATCACAGCCCAGGGAGTGCCTACTGCCTCGGA Hdac3.1037:(SEQ ID NO: 232) TGCTGTTGACAGTGAGCGCGAGGAACTTCCCTATAGTGAATAGTGAAGCCACAGATGTATTCACTATAGGGAAGTTCCTCATGCCTACTGCCTCGGA Hdac3.1491:(SEQ ID NO: 233) TGCTGTTGACAGTGAGCGCCCATATGTGGTTCTAGAATTATAGTGAAGCCACAGATGTATAATTCTAGAACCACATATGGTTGCCTACTGCCTCGGA Hdac3.506:(SEQ ID NO: 234) TGCTGTTGACAGTGAGCGATTCTGCTATGTCAATGACATATAGTGAAGCCACAGATGTATATGTCATTGACATAGCAGAAGTGCCTACTGCCTCGGA

shRNA Sequences for MEN1:

Men1.2310: (SEQ ID NO: 235)TGCTGTTGACAGTGAGCGCCACCCTCATCCTCTAATTCAATAGTGAAGCCACAGATGTATTGAATTAGAGGATGAGGGTGATGCCTACTGCCTCGGA Men1.219:(SEQ ID NO: 236) TGCTGTTGACAGTGAGCGACGGAATTGTAAGGAACTAGAATAGTGAAGCCACAGATGTATTCTAGTTCCTTACAATTCCGGTGCCTACTGCCTCGGA Men1.1457:(SEQ ID NO: 237) TGCTGTTGACAGTGAGCGACACTGTTATCCAAGACTACAATAGTGAAGCCACAGATGTATTGTAGTCTTGGATAACAGTGGTGCCTACTGCCTCGGA Men1.2707:(SEQ ID NO: 238) TGCTGTTGACAGTGAGCGACTGCCCGAATTTGGAAATCTTTAGTGAAGCCACAGATGTAAAGATTTCCAAATTCGGGCAGCTGCCTACTGCCTCGGA Men1.228:(SEQ ID NO: 239) TGCTGTTGACAGTGAGCGCAAGGAACTAGAAGGCCCTATATAGTGAAGCCACAGATGTATATAGGGCCTTCTAGTTCCTTATGCCTACTGCCTCGGA Men1.218:(SEQ ID NO: 62) TGCTGTTGACAGTGAGCGACCGGAATTGTAAGGAACTAGATAGTGAAGCCACAGATGTATCTAGTTCCTTACAATTCCGGGTGCCTACTGCCTCGGA

shRNA Sequences for SMARCA4

Smarca4.3232: (SEQ ID NO: 63)TGCTGTTGACAGTGAGCGAAAGGTAGAGTATGTCATCAAATAGTGAAGCCACAGATGTATTTGATGACATACTCTACCTTCTGCCTACTGCCTCGGA Smarca4.5466:(SEQ ID NO: 64) TGCTGTTGACAGTGAGCGCCTGGAGTCAGACAGTAATAAATAGTGAAGCCACAGATGTATTTATTACTGTCTGACTCCAGTTGCCTACTGCCTCGGA Smarca4.4935:(SEQ ID NO: 65) TGCTGTTGACAGTGAGCGACTCCGTCAAGGTGAAGATCAATAGTGAAGCCACAGATGTATTGATCTTCACCTTGACGGAGCTGCCTACTGCCTCGGA Smarca4.3364:(SEQ ID NO: 66) TGCTGTTGACAGTGAGCGCCTGATGAACACTATTATGCAATAGTGAAGCCACAGATGTATTGCATAATAGTGTTCATCAGTTGCCTACTGCCTCGGA Smarca4.3633:(SEQ ID NO: 67) TGCTGTTGACAGTGAGCGCCAGGCTTGATGGAACCACAAATAGTGAAGCCACAGATGTATTTGTGGTTCCATCAAGCCTGATGCCTACTGCCTCGGA Smarca4.4299:(SEQ ID NO: 68) TGCTGTTGACAGTGAGCGCCAGCGACTCACTGACAGAGAATAGTGAAGCCACAGATGTATTCTCTGTCAGTGAGTCGCTGTTGCCTACTGCCTCGGA

shRNA Sequences for SMARCD1

Smarcd1.986: (SEQ ID NO: 69)TGCTGTTGACAGTGAGCGAAAGCACTGTGGCAGTATATTATAGTGAAGCCACAGATGTATAATATACTGCCACAGTGCTTGTGCCTACTGCCTCGGA Smarcd1.1858:(SEQ ID NO: 70) TGCTGTTGACAGTGAGCGCTAGGACCTCTAGATAGTGTTATAGTGAAGCCACAGATGTATAACACTATCTAGAGGTCCTATTGCCTACTGCCTCGGA Smarcd1.690:(SEQ ID NO: 71) TGCTGTTGACAGTGAGCGCCGCGGCCTTGTCCAAATATGATAGTGAAGCCACAGATGTATCATATTTGGACAAGGCCGCGTTGCCTACTGCCTCGGA Smarcd1.1738:(SEQ ID NO: 72) TGCTGTTGACAGTGAGCGCCACCTGTTATCCCGTCCTGTATAGTGAAGCCACAGATGTATACAGGACGGGATAACAGGTGATGCCTACTGCCTCGGA Smarcd1.2668:(SEQ ID NO: 73) TGCTGTTGACAGTGAGCGACAGGTTTGTCACCCGGAGTTATAGTGAAGCCACAGATGTATAACTCCGGGTGACAAACCTGGTGCCTACTGCCTCGGA Smarcd1.1702:(SEQ ID NO: 74) TGCTGTTGACAGTGAGCGCCACAATGAAGAGGGTGTCACATAGTGAAGCCACAGATGTATGTGACACCCTCTTCATTGTGATGCCTACTGCCTCGGA

shRNA Sequences for SUZ12

Suz12.1676: (SEQ ID NO: 75)TGCTGTTGACAGTGAGCGATAGGATAGATGTTTCAATCAATAGTGAAGCCACAGATGTATTGATTGAAACATCTATCCTAGTGCCTACTGCCTCGGA Suz12.909:(SEQ ID NO: 76) TGCTGTTGACAGTGAGCGACTGGCAGTTTCCAGTAATGAATAGTGAAGCCACAGATGTATTCATTACTGGAAACTGCCAGGTGCCTACTGCCTCGGA Suz12.1842:(SEQ ID NO: 77) TGCTGTTGACAGTGAGCGATCGGAGTTTCTTGAATCTGAATAGTGAAGCCACAGATGTATTCAGATTCAAGAAACTCCGACTGCCTACTGCCTCGGA Suz12.3979:(SEQ ID NO: 78) TGCTGTTGACAGTGAGCGATAGGTGTAGAATTATTGCTTATAGTGAAGCCACAGATGTATAAGCAATAATTCTACACCTACTGCCTACTGCCTCGGA Suz12.4300:(SEQ ID NO: 79) TGCTGTTGACAGTGAGCGCTAAATGTTTATTTGAAATCAATAGTGAAGCCACAGATGTATTGATTTCAAATAAACATTTAATGCCTACTGCCTCGGA Suz12.419:(SEQ ID NO: 80) TGCTGTTGACAGTGAGCGACGCGGTGTTGCCGGTGAAGAATAGTGAAGCCACAGATGTATTCTTCACCGGCAACACCGCGGTGCCTACTGCCTCGGA

shRNA Sequences for WHSC111

Whsc111.1653: (SEQ ID NO: 81)TGCTGTTGACAGTGAGCGCACGAAGGGTATTGGTAACAAATAGTGAAGCCACAGATGTATTTGTTACCAATACCCTTCGTTTGCCTACTGCCTCGGA Whsc111.524:(SEQ ID NO: 82) TGCTGTTGACAGTGAGCGACTCACCCGAGATTAAACTAAATAGTGAAGCCACAGATGTATTTAGTTTAATCTCGGGTGAGCTGCCTACTGCCTCGGA Whsc111.276:(SEQ ID NO: 83) TGCTGTTGACAGTGAGCGCATCAGCTTGTATGAAACTCAATAGTGAAGCCACAGATGTATTGAGTTTCATACAAGCTGATTTGCCTACTGCCTCGGA Whsc111.373:(SEQ ID NO: 84) TGCTGTTGACAGTGAGCGCCTGACTATTACCATTCAGAAATAGTGAAGCCACAGATGTATTTCTGAATGGTAATAGTCAGTTGCCTACTGCCTCGGA Whsc111.1307:(SEQ ID NO: 85) TGCTGTTGACAGTGAGCGCTACCTCTAAGACGGAAGTCAATAGTGAAGCCACAGATGTATTGACTTCCGTCTTAGAGGTAATGCCTACTGCCTCGGA Whsc111.1059:(SEQ ID NO: 86) TGCTGTTGACAGTGAGCGCCGGGAATACAAAGGTCATGAATAGTGAAGCCACAGATGTATTCATGACCTTTGTATTCCCGTTGCCTACTGCCTCGGA

To confirm that inhibiting the genes identified in our screen would havetherapeutic potential when inhibited in vivo, subsequent validation wasperformed using a conditional, Tet-On RNAi vector (TRMPV) (FIG. 24).Using this approach, the mir30-embedded shRNA is cloned downstream ofthe Tet-responsive promoter (TRE). A downstream Pgk promoter drivesVenus and Neo-resistance cassette. To allow stable efficientTet-inducible activation, the rtTA3 (Tet-activator) gene is expressedfrom the same retroviral vector as the MLL-Af9 oncogene, separated by anIRES sequence. This manipulation of the retroviral vectors ensures thatall MLL-AF9 transformed cells will be competent for induction of theshRNA via doxycycline (a tetracycline analog) treatment. Each shRNAtargeting the epigenetic regulator is cloned into the TRMPV vector andintroduced into tet-on leukemia lines. Following neomycin selection, theleukemia cells are transplanted into recipient mice, which are thentreated with Doxycyline after day 3 of transplant (after the disease hasinitiated). Disease was monitored via bioluminescence, overall survivalbenefit, and relative contribution of dsRed+/shRNA+ cells to the finaldisease burden when the mice succumb to the disease.

This analysis was performed for the shRNAs targeting Eed, Aof2, Suz12,Men1, and Smarcd1, which revealed a survival benefit to mice, which forsome genes resulted in significant rates of cure in vivo (FIG. 25). Inaddition, all shRNAs inhibited proliferation of leukemia cells as thedisease developed, as compare to a Renilla luciferase control shRNA(FIG. 26). These findings validate that addictions of leukemia cells toepigenetic pathways, as identified by using an in vitro RNAi screen, canbe exploited for therapeutic benefit in vivo.

As an additional verification that therapy resistant AML leukemiasharbor a unique sensitivity to inhibition of the identified epigeneticpathways, the influence of the epigenetics shRNAs on normalhematopoiesis was also examined in vivo following retroviraltransduction of LMN (MSCV-miR30/shRNA-PGK-Neo-IRES-GFP; constructed byinserting a miR30-cassette and a PGK-NeomycinR-IRES-GFP cassette in theMSCV-backbone (Clontech)) into E13.5 fetal livers and subsequenttransplantation of infected cells into lethally irradiated recipients(FIG. 27). This assay compares the relative contribution of theexperimental shRNA vector (linked with GFP) to control, neutral shRNALMN vector that harbors the red fluorescent protein (mCherry). At 4weeks following transplantation of the mixed GFP+ and mCherry+ cellsinto recipient animals, the ratio of these two colors is measured insubsets of peripheral blood cells of various hematopoietic lineages orin bone marrow and spleens by co-staining with surface markers (e.g.Mac1, Gr1 for myeloid, B220 for B-lymphoid). Transduction with the Rpa3shRNA in the LMN-GFP vector is entirely depleted from normalhematopoietic cells after 4 weeks of transplantation. In contrast, theshRNAs targeting the epigenetic regulators Suz12.1676, Eed.1829, andSmarcd1.1858 have little, if any impact on proliferation of normalhematopoietic cells in vivo (FIG. 28). This approach furtherdemonstrates that the epigenetics RNAi screen has identified uniquehypersensitivities of the therapy-resistant AML leukemia cells.

5.12 Example 12 Use of Inhibitors of Epigenetic Gene Targets as NovelTherapies for Chemotherapy Resistant AML

An important feature of the proteins encoded by the gene targetsidentified from the above screen (Example 11) is their demonstratedenzymatic activity, identifying these proteins as attractive drugtargets. As a further aspect of this invention, such drug targets may beused in subsequent chemical screens to identify compounds able toinhibit their respective enzymatic activities. Compounds identifiedthrough such screening may provide for reasonable therapeutic strategiesin treatment of therapy-resistant AML leukemia.

As another aspect of this invention, known inhibitors of those proteinsare used for treatment of therapy-resistant AML leukemias. Compoundsuseful in the invention include Dznep and other Eed inhibitors,hydroximates (such as SAHA, TSA and CBHA) and other HDAC inhibitors, andMAO-inhibitors and other inhibitors with activity against Aof2(lysine-specific demethylase-1/LSD1). (Tan J et al., Genes Dev. 2007 May1; 21(9):1050-63, Lane A A et al., J Clin Oncol. 2009 Nov. 10;27(32):5459-68, Miller et al., J Med. Chem. 2003; 46: 5097-5116, Lee M Get al., Chem Biol. 2006 June; 13(6):563-7, Ueda R et. al., J Am. Chem.Soc. 2009 Dec. 9; 131(48):17536-7).

As an initial investigation of the applicability of these compounds intherapy-resistant AML leukemia, we tested whether tranylcypromine (anFDA-approved MAO inhibitor that has inhibitory activity towards Aof2)impacted proliferation of MLL-leukemia cells in vitro. (FIG. 29). Weobserved a dose-dependent inhibition of MLL-leukemia cell proliferationupon exposing leukemia cells to tranylcypromine. In contrast, exposureto tranylcypromine had no impact on growth of the non-transformed 32Dcell line. We also observed an effect on growth of several human AMLcell lines (HL60, Kasumi1, Molm13), but not others (KG1). These findingsindicate that use of small molecules that target the epigenetic enzymesidentified in our screen may provide reasonable therapeutic strategiesfor this otherwise therapy-resistant disease.

5.13 Example 13 Screening and Evaluation of RNAi Targets by PooledNegative Selection RNAi Screening In Vitro and In Vivo

In addition to the analysis of single shRNAs for inhibitory effects invitro and in vivo, the combination of tet-on competent cancer models andtet-regulatable shRNA expression vectors allowing for monitoring shRNAexpression through fluorescent and other reporter genes (e.g. TRMPV andderivates) facilitates pooled shRNA negative selection screening (FIG.37). In such approaches a pool of over 1,000 shRNAs are retrovirallytransduced into tet-on competent cancer models (here MLL/AF9+Nras AML)and selected for shRNA containing cells by either drug selection (e.g.G418) or fluorescence-activated cell sorting (FACS). Selected cellpopulations harboring a library of shRNAs then can be cultured in theabsence or presence of doxycyline (off dox and on dox, respectively) orinjected into syngeneic recipient mice that are either treated withdoxycyline or left untreated.

The representation of each shRNA within the pool can be effectivelyassessed by deep sequencing of shRNA cassettes in a given cellpopulation. For this, genomic DNA is extracted from leukemia cellscontaining the shRNA pool, and the shRNA guide or passenger strand(21-22 target gene specific nucleotides of the shRNA) by PCR usingprimers specific for common sequences flanking the gene specific part ofthe shRNA (e.g. miR30 19 nt loop and miR30 common 3′ sequence). Theprimers also contain 5′-adapters required for deep sequencing (e.g. forIllumina/Solexa sequencing primers are p5+mir3:5′-AATGATACGGCGACCACCGACTAAAGTAGCCCCTTGAATTC-3′ (SEQ ID NO: 215) andp7+Loop: 5′-CAAGCAGAAGACGGCATACGATAGTGAAGCCACAGATGTA-3′) (SEQ ID NO:216). Deep sequencing is subsequently performed using a primer bindingthe flanking nucleotide sequence next to the target gene specific partof the shRNA (for Illumina/Solexa platforms the primer is mir30EcoR1Seq:5′-TAGCCCCTTGAATTCCGA GGCAGTAGGCA-3′ (SEQ ID NO: 217)).

The use of shRNA vectors that allow for monitoring of shRNA expressionthrough fluorescent and other reporter genes (e.g. TRMPV) in combinationwith tet-on competent mouse models facilitates the isolation of pureshRNA expressing cell populations (e.g. by FACS, for TRMPV by sorting ofVenus/dsRed double positive cells (FIG. 38). This selection of shRNAexpressing cells prior to DNA isolation strongly reduces the backgroundcaused by cell populations with insufficient shRNA induction, which arepredicted to contain genomic representation of the whole shRNA librarywithout shRNA specific shifts in representation. The final readout isbased on the comparison of shRNA representation (i.e. deep sequencingread numbers) before the assay (t0) or from cell populations leftwithout doxycycline treatment (off dox) to those where shRNA wereinduced and cells subsequently sorted for shRNA expressing cells (ondox). shRNA with inhibitory effects are predicted to looserepresentation (show less reads) on dox compared to t0 and off dox.

This pooled is RNAi screening strategy can be applied to rapidly surveyinhibitory effects of large pools of shRNAs (˜1000 at a time) indifferent tet-on competent cancer models and normal control cells (FIG.39). For example, a pool of 1166 shRNAs targeting 836 predominantlydruggable genes associated with MLL fusion proteins in human leukemiaand mouse models of MLL/AF9-induced AML (MLL cure library) was analyzedin pooled negative selection screening in tet-on competent MLL/AF9+NrasAML and immortalized murine embryonic fibroblasts (Mef) in parallel. Toestablish and validate this screening approach, the pool also contained64 control shRNAs each of which have been previously analyzed forinhibitory effects in both AML cells and Mefs using the sameexperimental system (FIG. 40).

After pooling of 1166 experimental and 64 control shRNAs the presence ofshRNAs was verified by deep sequencing of shRNAs within the plasmidvector pool (FIG. 41). The library of 1230 shRNAs was then retrovirallytransduced into target cells at predominantly single copy integration(data not shown) into approximately 1000 target cells for each shRNA(1000 fold library representation). The successful transduction of thewhole shRNA library was confirmed by deep sequencing from infected cellsfollowing selection, which demonstrated a high correlation of shRNAreads in the plasmid vector pool and the infected/selected cellpopulation (FIG. 42).

In addition we demonstrate that the library representation can bemaintained during prolonged periods of passaging in cell culture (FIGS.43-44) as well as after transplantation into syngeneic recipient mice(FIGS. 45-46). Furthermore, independent biological replicates of on doxsamples show a high correlation of shRNA representation both in vitroand in vivo indicating that shRNA-mediated effects and consequentchanges in library representation are specific and not random (FIGS.47-48).

After analysis of deep sequencing a total of 119 shRNAs wereconsistently found more than 8-fold depleted in MLL/AF9+Nras induced AMLin independent biological replicates in vitro and in vivo. None of the44 neutral control shRNAs showed this level of depletion (FIG. 49),while 18 out of 22 inhibitory control shRNAs were found more than 8 foldreduced in representation (FIGS. 50-51). Importantly, for each controlgene known to be required for maintenance of MLL/AF9+Nras leukemia(Rpa1, Rpa3, PCNA, Myc, Myb, Bcl2, Mcl1 and Telo2) at least, mostlymultiple shRNAs were identified as more than 8-fold depleted indicatingthat this pooled screening approach can efficiently and specificallyidentify genes required for cancer cell survival. Out of 101 depletednon-control shRNAs 88 showed specific depletion in MLL/AF9+Nras leukemiaand were not significantly altered in Mefs.

Thus, these shRNAs point out genes that are specifically required inMLL/AF9+Nras leukemia maintenance, but dispensable for survival ofnormal Mef cells. These genes are Acpp, Acs11, Adam23, Ap1s2, Arf3,Aspa, Atp6v0d1, AU018778, Cacna1f, Casp1, Ccr1, Cd6, Cdc42ep1, Cdc42ep3,Centa1, Centd3, Cerk, Cpd, Ctbs, Cx3cr1, Dio2, Dnajc10, Dsp, EG277089,EG408196, F10, Fas, Fgd4, Flot2, Fn1, Fn3k, Fosb, Fpr1, Gab3, Gart,Gas1, Gdi1, Hexa, Hpgd, Htatip2, Kcnh7, Klf5, Klrb1b, L1cam, Lima1,Mef2c, Mgst1, Myo7a, Ncf1, Nln, Nmur1, Nrg4, Nrp1, Ntrk3, Ogg1, Park2,Pctk1, Pde1b, Pdgfrb, Pgam1, Pitpnm1, Pkm2, Plcb2, Plod3, Ptpn18, Pyg1,Rbks, Rgs6, Rock2, S100a9, Slc11a1, Slc12a5, Slc15a3, Slc22a4, Slc6a13,Smpd2, Syt17, Tex14, Thyn1, Tnfsf12, Trpm4, Vnn3 and Wnt10b.

TABLE 2 shRNAs specifically depleted in MLL/AF9+Nras leukemia cells ascompared to Mef cells (shRNAs specifically inhibiting MLL/AF9 inducedAML, but not Mefs) Fold depletion Fold depletion Fold depletion Folddepletion shRNA in AML in Mef shRNA in AML in Mef Hexa.1510 1,777.362.13 EG277089.605 25.37 2.02 Ccr1.733 472.15 1.18 Gdi1.1120 24.81 1.98Cdc42ep3.1922 315.30 0.38 Plod3.3138 21.28 0.71 Dsp.6155 242.96 0.60EG408196.85 20.79 0.25 Gart.2470 146.15 0.67 Trpm4.2813 20.52 2.33Thyn1.315 145.53 1.04 Dio2.2087 20.52 2.52 Gas1.2159 132.34 0.81Pgam1.1811 20.18 0.17 Pctk1.948 123.76 0.27 Nmur1.7 19.45 2.58 Pygl.1463118.65 1.94 Ctbs.796 19.44 0.19 Fn3k.298 103.54 2.82 Rbks.337 17.02 1.07Arf3.1357 94.96 0.86 Lima1.3235 16.44 0.56 Mef2c.1872 63.15 0.77Casp1.248 16.16 3.84 Pitpnm1.561 60.72 0.27 Pdgfrb.1028 16.16 2.15Ap1s2.2744 57.84 2.31 Htatip2.575 15.74 1.08 Cpd.1693 50.07 3.91Wnt10b.1717 15.69 1.35 Slc22a4.2223 41.21 3.41 Pde1b.2027 15.12 1.31Adam23.5211 40.11 0.30 Cdc42ep1.317 14.91 1.47 Fn1.4143 36.54 0.84Myo7a.618 14.79 4.42 Tnfsf12.1204 36.03 0.93 Rgs6.896 14.33 1.99L1cam.5255 31.42 1.26 Nrp1.2022 13.60 1.80 Cd6.520 28.66 2.28Centd3.3993 13.37 1.77 Cerk.4466 27.79 0.61 Aspa.996 13.25 0.68 Fgd4.82125.66 2.12 S100a9.148 13.18 2.68 Fosb.1165 13.12 3.61 Klrb1b.845 9.740.43 Cx3cr1.1223 12.98 0.69 Fpr1.375 9.66 3.07 Slc12a5.3582 12.58 2.21Pkm2.2154 9.63 1.92 Cacna1f.3314 12.52 2.14 Tex14.3976 9.60 1.90Plod3.2501 12.30 2.62 Slc11a1.307 9.52 0.41 Atp6vOd1.481 12.03 1.90Klf5.581 9.37 1.01 Centa1.1387 11.44 0.77 Mgst1.549 9.14 1.09 Park2.222211.36 1.32 Acpp.818 9.00 0.85 Flot2.1039 10.89 2.72 Dnajc10.2057 8.991.47 Plcb2.3742 10.81 2.25 Ntrk3.167 8.88 1.76 Kcnh7.2642 10.58 0.92Gart.2713 8.73 2.22 F10.1044 10.52 1.25 Ogg1.395 8.69 1.44 Hpgd.107010.43 1.10 Smpd2.592 8.63 2.89 Slc15a3.1086 10.29 2.32 Fas.1344 8.571.36 Syt17.721 10.25 0.86 AU018778.350 8.45 1.18 Gab3.527 10.22 0.46Centa1.940 8.24 1.13 Vnn3.345 10.19 2.59 Nrp1.479 8.19 0.83 Acsl1.355910.18 1.45 Ptpn18.247 8.13 0.21 Ncf1.2370 10.06 2.33 Nrg4.1358 8.11 0.54Gdi1.2355 9.92 2.86 Rock2.3898 8.10 1.04 Slc6a13.1196 9.77 1.52 Nln.14298.03 1.61

TABLE 3 MLL/AF9 specific control shRNAs (shRNAs inhibiting MLL/AF9induced AML, but not Mefs) shRNA Fold depletion in AML Fold depletion inMef Bcl2.1422 1,686.19 2.71 Bcl2.2169 12.61 1.62 Bcl2.906 12.28 0.98Mel1.1334 104.03 3.00 Mcl1.2018 11.48 3.02 Myb.2572 67.47 0.92 Myb.265215.46 1.14 Myb.670 26.46 1.28

TABLE 4 shRNAs inihibiting both MLL/AF9 induced AML and Mefs. shRNA Folddepletion in AML Fold depletion in Mef Myc.1888 8.48 3.83 Myc.1891 9.054.62 Myc.2105 9.19 11.70 Pcna.1186 51.94 11.42 Pcna.566 9.89 6.15Rpa1.1620 2,891.47 10.23 Rpa3.561 8.81 10.63

TABLE 5 Neutral control shRNAs (functional shRNAs that do not inhibitMLL/AF9 induced AML and Mefs) shRNA Fold depletion in AML Fold depletionin Mef BRAF.3750 0.76 1.08 BRAF.3826 0.92 0.87 BRAF.5053 0.66 0.33Kit.1241 1.88 0.99 Kit.2021 1.92 1.61 Kit.221 1.19 0.69 Kit.4813 0.810.84 Lin28.2180 1.15 0.80 Lin28.2186 1.43 1.55 Lin28.2270 0.22 1.10Lin28.2430 1.37 0.89 Luciferase.1309 1.22 0.52 Map2k1.1200 2.26 1.24Map2k1.2337 0.24 0.68 Mn1.1403 1.94 0.77 Mn1.2545 0.81 0.29 Mn1.57600.38 0.91 Mn1.5864 0.61 1.09 Ptgs2.1082 0.51 0.51 Ptgs2.2058 1.78 1.27Ptgs2.284 0.34 1.21 Ptgs2.3711 1.38 1.46 Renilla.713 1.29 1.02Trp53.1224 0.10 0.84

The shRNAs of Table 2-4, identified as having potent antileukemiceffects, and in particular specific antileukemic effects (Tables 2-3)are directly useful in various embodiments of the invention.Additionally, it should be appreciated that modified shRNA molecules andsiRNA molecules directed against the targeted genes and derived fromthese shRNAs according to the disclosures herein (for example, asexemplified in sections 4.3-4.5, Example 3) are useful in otherembodiments of the invention, and in particular, in the therapeutic useof such siRNA molecules to treat therapy resistant cancers.

The sequences of the shRNAs listed in Tables 2-4 are listed below as theoligo sequence:

Acpp.818: (SEQ ID NO: 87)TGCTGTTGACAGTGAGCGAAAAGAGAAATCTCGACTCCAATAGTGAAGCCACAGATGTATTGGAGTCGAGATTTCTCTTTCTGCCTACTGCCTCGGA Acsl1.3559:(SEQ ID NO: 88) TGCTGTTGACAGTGAGCGCCAGCATTTCACTTTACTGCAATAGTGAAGCCACAGATGTATTGCAGTAAAGTGAAATGCTGTTGCCTACTGCCTCGGA Adam23.5211:(SEQ ID NO: 89) TGCTGTTGACAGTGAGCGCTACGACCACGTCAGTTACAAATAGTGAAGCCACAGATGTATTTGTAACTGACGTGGTCGTATTGCCTACTGCCTCGGA Apls2.2744:(SEQ ID NO: 90) TGCTGTTGACAGTGAGCGACAGGAGCAAGATGAGTTACTATAGTGAAGCCACAGATGTATAGTAACTCATCTTGCTCCTGCTGCCTACTGCCTCGGA Arf3.1357:(SEQ ID NO: 91) TGCTGTTGACAGTGAGCGACCCTTCTGTGTTGGTGAGATATAGTGAAGCCACAGATGTATATCTCACCAACACAGAAGGGCTGCCTACTGCCTCGGA Aspa.996:(SEQ ID NO: 92) TGCTGTTGACAGTGAGCGCATGAAGCTGCATATTATGAAATAGTGAAGCCACAGATGTATTTCATAATATGCAGCTTCATTTGCCTACTGCCTCGGA Atp6v0d1.481:(SEQ ID NO: 93) TGCTGTTGACAGTGAGCGACACCAGCGTTCAATAGCTGAATAGTGAAGCCACAGATGTATTCAGCTATTGAACGCTGGTGCTGCCTACTGCCTCGGA AU018778.350:(SEQ ID NO: 94) TGCTGTTGACAGTGAGCGCCTGAAGATTGCCTGTACCTAATAGTGAAGCCACAGATGTATTAGGTACAGGCAATCTTCAGATGCCTACTGCCTCGGA Bcl2.1422:(SEQ ID NO: 95) TGCTGTTGACAGTGAGCGACCGGGAGAACAGGGTATGATATAGTGAAGCCACAGATGTATATCATACCCTGTTCTCCCGGCTGCCTACTGCCTCGGA Bcl2.2169:(SEQ ID NO: 96) TGCTGTTGACAGTGAGCGACAGTAGAAATTATATGCATTATAGTGAAGCCACAGATGTATAATGCATATAATTTCTACTGCTGCCTACTGCCTCGGA Bcl2.757:(SEQ ID NO: 97) TGCTGTTGACAGTGAGCGCCCCGATTCATTGCAAGTTGTATAGTGAAGCCACAGATGTATACAACTTGCAATGAATCGGGATGCCTACTGCCTCGGA Bcl2.906:(SEQ ID NO: 98) TGCTGTTGACAGTGAGCGCGCACAGGAATTTTGTTTAATATAGTGAAGCCACAGATGTATATTAAACAAAATTCCTGTGCATGCCTACTGCCTCGGA Cacna1f.3314:(SEQ ID NO: 99) TGCTGTTGACAGTGAGCGACTGGCCTGCGCTACTATACAATAGTGAAGCCACAGATGTATTGTATAGTAGCGCAGGCCAGCTGCCTACTGCCTCGGA Casp1.248:(SEQ ID NO: 100) TGCTGTTGACAGTGAGCGCCAGTGAGTATAGGGACAATAATAGTGAAGCCACAGATGTATTATTGTCCCTATACTCACTGATGCCTACTGCCTCGGA Ccr1.733:(SEQ ID NO: 101) TGCTGTTGACAGTGAGCGCCTGGATTGACTACAAGTTGAATAGTGAAGCCACAGATGTATTCAACTTGTAGTCAATCCAGATGCCTACTGCCTCGGA Cd6.520:(SEQ ID NO: 102) TGCTGTTGACAGTGAGCGAGAGCCACTTCTGGGAACACAATAGTGAAGCCACAGATGTATTGTGTTCCCAGAAGTGGCTCCTGCCTACTGCCTCGGA Cdc42ep1.317:(SEQ ID NO: 103) TGCTGTTGACAGTGAGCGCCTAGCAGTTGTAAGCAATCAATAGTGAAGCCACAGATGTATTGATTGCTTACAACTGCTAGTTGCCTACTGCCTCGGA Cdc42ep3.1922:(SEQ ID NO: 104) TGCTGTTGACAGTGAGCGCAACAAGCAAGGTATTACTGTATAGTGAAGCCACAGATGTATACAGTAATACCTTGCTTGTTTTGCCTACTGCCTCGGA Centa1.1387:(SEQ ID NO: 105) TGCTGTTGACAGTGAGCGCCAGAACCTCATTAAAGTTGAATAGTGAAGCCACAGATGTATTCAACTTTAATGAGGTTCTGATGCCTACTGCCTCGGA Centa1.940:(SEQ ID NO: 106) TGCTGTTGACAGTGAGCGCCCGAAGGCTCATGTACTTCAATAGTGAAGCCACAGATGTATTGAAGTACATGAGCCTTCGGTTGCCTACTGCCTCGGA Centd3.3993:(SEQ ID NO: 107) TGCTGTTGACAGTGAGCGCCTGGACCACAAGCATCCTTAATAGTGAAGCCACAGATGTATTAAGGATGCTTGTGGTCCAGTTGCCTACTGCCTCGGA Cerk.4466:(SEQ ID NO: 108) TGCTGTTGACAGTGAGCGCCTGGTATATTTGAGAAGACAATAGTGAAGCCACAGATGTATTGTCTTCTCAAATATACCAGATGCCTACTGCCTCGGA Cpd.1693:(SEQ ID NO: 109) TGCTGTTGACAGTGAGCGCACCAGGTGAACCAGAATTTAATAGTGAAGCCACAGATGTATTAAATTCTGGTTCACCTGGTTTGCCTACTGCCTCGGA Ctbs.796:(SEQ ID NO: 110) TGCTGTTGACAGTGAGCGCCCCACAGAGCATCTCACTAAATAGTGAAGCCACAGATGTATTTAGTGAGATGCTCTGTGGGTTGCCTACTGCCTCGGA Cx3cr1.1223:(SEQ ID NO: 111) TGCTGTTGACAGTGAGCGACTGCATCTTATGTGCAAGAAATAGTGAAGCCACAGATGTATTTCTTGCACATAAGATGCAGGTGCCTACTGCCTCGGA Dio2.2087:(SEQ ID NO: 112) TGCTGTTGACAGTGAGCGCCAGGAATTTGGTTAAATGGAATAGTGAAGCCACAGATGTATTCCATTTAACCAAATTCCTGTTGCCTACTGCCTCGGA Dnajc10.2057:(SEQ ID NO: 113) TGCTGTTGACAGTGAGCGCTCCAACGACAGTGGTATTCAATAGTGAAGCCACAGATGTATTGAATACCACTGTCGTTGGATTGCCTACTGCCTCGGA Dsp.6155:(SEQ ID NO: 114) TGCTGTTGACAGTGAGCGCCAGGAAGTTCTTCGATCAATATAGTGAAGCCACAGATGTATATTGATCGAAGAACTTCCTGTTGCCTACTGCCTCGGA EG277089.605:(SEQ ID NO: 115) TGCTGTTGACAGTGAGCGCCTCAATCTCATTGGTGGCTTATAGTGAAGCCACAGATGTATAAGCCACCAATGAGATTGAGATGCCTACTGCCTCGGA EG408196.85:(SEQ ID NO: 116) TGCTGTTGACAGTGAGCGCAGCGAGAACCAGTGAGAAATATAGTGAAGCCACAGATGTATATTTCTCACTGGTTCTCGCTTTGCCTACTGCCTCGGA F10.1044:(SEQ ID NO: 117) TGCTGTTGACAGTGAGCGACACCATCTTGAATGAGTTCTATAGTGAAGCCACAGATGTATAGAACTCATTCAAGATGGTGCTGCCTACTGCCTCGGA Fas.1344:(SEQ ID NO: 118) TGCTGTTGACAGTGAGCGCGAGGAGAATTATAAACTGAAATAGTGAAGCCACAGATGTATTTCAGTTTATAATTCTCCTCATGCCTACTGCCTCGGA Fgd4.821:(SEQ ID NO: 119) TGCTGTTGACAGTGAGCGAAAGGAGACTAATGAACAGAAATAGTGAAGCCACAGATGTATTTCTGTTCATTAGTCTCCTTCTGCCTACTGCCTCGGA Flot2.1039:(SEQ ID NO: 120) TGCTGTTGACAGTGAGCGACAAGGTGACATCAGAAGTAAATAGTGAAGCCACAGATGTATTTACTTCTGATGTCACCTTGCTGCCTACTGCCTCGGA Fn1.4143:(SEQ ID NO: 121) TGCTGTTGACAGTGAGCGCCAGTAGGATACTACACAGTTATAGTGAAGCCACAGATGTATAACTGTGTAGTATCCTACTGATGCCTACTGCCTCGGA Fn3k.298:(SEQ ID NO: 122) TGCTGTTGACAGTGAGCGAAAGAGCCTTAGCAGTCAGGCATAGTGAAGCCACAGATGTATGCCTGACTGCTAAGGCTCTTCTGCCTACTGCCTCGGA Fosb.1165:(SEQ ID NO: 123) TGCTGTTGACAGTGAGCGCCAGGCGGAAACTGATCAGCTTTAGTGAAGCCACAGATGTAAAGCTGATCAGTTTCCGCCTGATGCCTACTGCCTCGGA Fpr1.375:(SEQ ID NO: 124) TGCTGTTGACAGTGAGCGATTGGTTCATGTGCAAATTCATTAGTGAAGCCACAGATGTAATGAATTTGCACATGAACCAACTGCCTACTGCCTCGGA Gab3.527:(SEQ ID NO: 125) TGCTGTTGACAGTGAGCGCGACGGAAACACTAATAGTGTATAGTGAAGCCACAGATGTATACACTATTAGTGTTTCCGTCATGCCTACTGCCTCGGA Gart.2470:(SEQ ID NO: 126) TGCTGTTGACAGTGAGCGCAAGAATCTGATTGAAACCATATAGTGAAGCCACAGATGTATATGGTTTCAATCAGATTCTTATGCCTACTGCCTCGGA Gart.2713:(SEQ ID NO: 127) TGCTGTTGACAGTGAGCGAACCAGGGTAATTAATCACAAATAGTGAAGCCACAGATGTATTTGTGATTAATTACCCTGGTGTGCCTACTGCCTCGGA Gas1.2159:(SEQ ID NO: 128) TGCTGTTGACAGTGAGCGACCCGAAATTACAACTGCATTATAGTGAAGCCACAGATGTATAATGCAGTTGTAATTTCGGGGTGCCTACTGCCTCGGA Gdi1.1120:(SEQ ID NO: 129) TGCTGTTGACAGTGAGCGCCAACAGGAAGTCAGACATCTATAGTGAAGCCACAGATGTATAGATGTCTGACTTCCTGTTGATGCCTACTGCCTCGGA Gdi1.2355:(SEQ ID NO: 130) TGCTGTTGACAGTGAGCGACTCTAGTATATTTCACAGAAATAGTGAAGCCACAGATGTATTTCTGTGAAATATACTAGAGCTGCCTACTGCCTCGGA Hexa.1510:(SEQ ID NO: 131) TGCTGTTGACAGTGAGCGAGAGCAGTAACCTGACAACTAATAGTGAAGCCACAGATGTATTAGTTGTCAGGTTACTGCTCCTGCCTACTGCCTCGGA Hpgd.1070:(SEQ ID NO: 132) TGCTGTTGACAGTGAGCGCAAACTAGGTTATAACCTATAATAGTGAAGCCACAGATGTATTATAGGTTATAACCTAGTTTTTGCCTACTGCCTCGGA Htatip2.575:(SEQ ID NO: 133) TGCTGTTGACAGTGAGCGACAGCAGTTTCTTATACCTACATAGTGAAGCCACAGATGTATGTAGGTATAAGAAACTGCTGGTGCCTACTGCCTCGGA Kcnh7.2642:(SEQ ID NO: 134) TGCTGTTGACAGTGAGCGCATGGTTCATCTTTATGCCAAATAGTGAAGCCACAGATGTATTTGGCATAAAGATGAACCATTTGCCTACTGCCTCGGA Klf5.581:(SEQ ID NO: 135) TGCTGTTGACAGTGAGCGCTCCGATAATTTCAGAGCATAATAGTGAAGCCACAGATGTATTATGCTCTGAAATTATCGGAATGCCTACTGCCTCGGA Klrb1b.845:(SEQ ID NO: 136) TGCTGTTGACAGTGAGCGCCAGATTCTTCATTGTATAAATTAGTGAAGCCACAGATGTAATTTATACAATGAAGAATCTGTTGCCTACTGCCTCGGA L1cam.5255:(SEQ ID NO: 137) TGCTGTTGACAGTGAGCGCCAGAATTATAACAGGCAAATATAGTGAAGGCCACAGATGTATATTTGCCTGTTATAATTCTGATGCCTACTGCCTCGGA Lima1.3235:(SEQ ID NO: 138) TGCTGTTGACAGTGAGCGAACGGACATTGTACCCAGATAATAGTGAAGCCACAGATGTATTATCTGGGTACAATGTCCGTCTGCCTACTGCCTCGGA Mcl1.1334:(SEQ ID NO: 139) TGCTGTTGACAGTGAGCGAAAGAGTCACTGTCTGAATGAATAGTGAAGCCACAGATGTATTCATTCAGACAGTGACTCTTCTGCCTACTGCCTCGGA Mcl1.2018:(SEQ ID NO: 140) TGCTGTTGACAGTGAGCGCGGACTGGTTATAGATTTATAATAGTGAAGCCACAGATGTATTATAAATCTATAACCAGTCCATGCCTACTGCCTCGGA Mef2c.1872:(SEQ ID NO: 141) TGCTGTTGACAGTGAGCGCTGCCTCAGTGATACAGTATAATAGTGAAGCCACAGATGTATTATACTGTATCACTGAGGCAATGCCTACTGCCTCGGA Mgst1.549:(SEQ ID NO: 142) TGCTGTTGACAGTGAGCGCAAGGAGCAGACTGTACTTGTATAGTGAAGCCACAGATGTATACAAGTACAGTCTGCTCCTTATGCCTACTGCCTCGGA Myb.2572:(SEQ ID NO: 143) TGCTGTTGACAGTGAGCGCTCCATGTATCTCAGTCACTAATAGTGAAGCCACAGATGTATTAGTGACTGAGATACATGGAATGCCTACTGCCTCGGA Myb.2652:(SEQ ID NO: 144) TGCTGTTGACAGTGAGCGCCCCAAGTAATACTTAATGCAATAGTGAAGCCACAGATGTATTGCATTAAGTATTACTTGGGATGCCTACTGCCTCGGA Myb.670:(SEQ ID NO: 145) TGCTGTTGACAGTGAGCGACACAACCATTTGAATCCAGAATAGTGAAGCCACAGATGTATTCTGGATTCAAATGGTTGTGCTGCCTACTGCCTCGGA Myc.1888:(SEQ ID NO: 146) TGCTGTTGACAGTGAGCGAGAAACGACGAGAACAGTTGAATAGTGAAGCCACAGATGTATTCAACTGTTCTCGTCGTTTCCTGCCTACTGCCTCGGA Myc.1891:(SEQ ID NO: 147) TGCTGTTGACAGTGAGCGCACGACGAGAACAGTTGAAACATAGTGAAGCCACAGATGTATGTTTCAACTGTTCTCGTCGTTTGCCTACTGCCTCGGA Myc.2105:(SEQ ID NO: 148) TGCTGTTGACAGTGAGCGCCTGCCTCAAACTTAAATAGTATAGTGAAGCCACAGATGTATACTATTTAAGTTTGAGGCAGTTGCCTACTGCCTCGGA Myo7a.618:(SEQ ID NO: 149) TGCTGTTGACAGTGAGCGCCCGCCAGTACACCAACAAGAATAGTGAAGCCACAGATGTATTCTTGTTGGTGTACTGGCGGATGCCTACTGCCTCGGA Ncf1.2370:(SEQ ID NO: 150) TGCTGTTGACAGTGAGCGCCAGAAGATCAATGCACATAAATAGTGAAGCCACAGATGTATTTATGTGCATTGATCTTCTGTTGCCTACTGCCTCGGA Nln.1429:(SEQ ID NO: 151) TGCTGTTGACAGTGAGCGAAAGGATAAAGCTACTGGAGAATAGTGAAGCCACAGATGTATTCTCCAGTAGCTTTATCCTTCTGCCTACTGCCTCGGA Nmur1.7:(SEQ ID NO: 152) TGCTGTTGACAGTGAGCGCCTGCAATATCAGTGAGTTCAATAGTGAAGCCACAGATGTATTGAACTCACTGATATTGCAGATGCCTACTGCCTCGGA Nrg4.1358:(SEQ ID NO: 153) TGCTGTTGACAGTGAGCGCCAGACATGTTGAAGTGAATAATAGTGAAGCCACAGATGTATTATTCACTTCAACATGTCTGTTGCCTACTGCCTCGGA Nrp1.2022:(SEQ ID NO: 154) TGCTGTTGACAGTGAGCGACACAAGGTTCATCAGGATCTATAGTGAAGCCACAGATGTATAGATCCTGATGAACCTTGTGGTGCCTACTGCCTCGGA Nrp1.479:(SEQ ID NO: 155) TGCTGTTGACAGTGAGCGCACCCTCATTCTTACCATCCAATAGTGAAGCCACAGATGTATTGGATGGTAAGAATGAGGGTATGCCTACTGCCTCGGA Ntrk3.167:(SEQ ID NO: 156) TGCTGTTGACAGTGAGCGCCTGCAGCAAGACTGAGATCAATAGTGAAGCCACAGATGTATTGATCTCAGTCTTGCTGCAGATGCCTACTGCCTCGGA Ogg1.395:(SEQ ID NO: 157) TGCTGTTGACAGTGAGCGACAGATCAAGTATGGACACTGATAGTGAAGCCACAGATGTATCAGTGTCCATACTTGATCTGCTGCCTACTGCCTCGGA Park2.2222:(SEQ ID NO: 158) TGCTGTTGACAGTGAGCGCAACAGAGAAAGTGCCTATAAATAGTGAAGCCACAGATGTATTTATAGGCACTTTCTCTGTTATGCCTACTGCCTCGGA Pcna.1186:(SEQ ID NO: 159) TGCTGTTGACAGTGAGCGAATCAATGATCTTGACGCTAAATAGTGAAGCCACAGATGTATTTAGCGTCAAGATCATTGATGTGCCTACTGCCTCGGA Pcna.566:(SEQ ID NO: 160) TGCTGTTGACAGTGAGCGATCGGGTGAATTTGCACGTATATAGTGAAGCCACAGATGTATATACGTGCAAATTCACCCGACTGCCTACTGCCTCGGA Pctk1.948:(SEQ ID NO: 161) TGCTGTTGACAGTGAGCGCCCGGGAAGTATCCCTGCTTAATAGTGAAGCCACAGATGTATTAAGCAGGGATACTTCCCGGATGCCTACTGCCTCGGA Pde1b.2027:(SEQ ID NO: 162) TGCTGTTGACAGTGAGCGCTGCCTCCAAGTTTCTAAGCAATAGTGAAGCCACAGATGTATTGCTTAGAAACTTGGAGGCAATGCCTACTGCCTCGGA Pdgfrb.1028:(SEQ ID NO: 163) TGCTGTTGACAGTGAGCGAAACGACCATGGCGATGAGAAATAGTGAAGCCACAGATGTATTTCTCATCGCCATGGTCGTTCTGCCTACTGCCTCGGA Pgam1.1811:(SEQ ID NO: 164) TGCTGTTGACAGTGAGCGCAAGGAGTGATGTGCAATACTTTAGTGAAGCCACAGATGTAAAGTATTGCACATCACTCCTTTTGCCTACTGCCTCGGA Pitpnm1.561:(SEQ ID NO: 165) TGCTGTTGACAGTGAGCGCCAGGATGCTTATCAAGGAGTATAGTGAAGCCACAGATGTATACTCCTTGATAAGCATCCTGATGCCTACTGCCTCGGA Pkm2.2154:(SEQ ID NO: 166) TGCTGTTGACAGTGAGCGCGCCCACCTGAATGTCAATAAATAGTGAAGCCACAGATGTATTTATTGACATTCAGGTGGGCATGCCTACTGCCTCGGA Plcb2.3742:(SEQ ID NO: 167) TGCTGTTGACAGTGAGCGCAGGGACCTTAATACTCAGATATAGTGAAGCCACAGATGTATATCTGAGTATTAAGGTCCCTTTGCCTACTGCCTCGGA Plod3.2501:(SEQ ID NO: 168) TGCTGTTGACAGTGAGCGAACCGTTGATATCCACATGAAATAGTGAAGCCACAGATGTATTTCATGTGGATATCAACGGTGTGCCTACTGCCTCGGA Plod3.3138:(SEQ ID NO: 169) TGCTGTTGACAGTGAGCGACTCAGCCTCACTTTCAATAAATAGTGAAGCCACAGATGTATTTATTGAAAGTGAGGCTGAGGTGCCTACTGCCTCGGA Ptpn18.247:(SEQ ID NO: 170) TGCTGTTGACAGTGAGCGCCACGAACAAGAACCGCTACAATAGTGAAGCCACAGATGTATTGTAGCGGTTCTTGTTCGTGTTGCCTACTGCCTCGGA Pyg1.1463:(SEQ ID NO: 171) TGCTGTTGACAGTGAGCGCACCAGACAAGTTCCAGAATAATAGTGAAGCCACAGATGTATTATTCTGGAACTTGTCTGGTTTGCCTACTGCCTCGGA Rbks.337:(SEQ ID NO: 172) TGCTGTTGACAGTGAGCGAAGCCTCCATAATTGTCAATAATAGTGAAGCCACAGATGTATTATTGACAATTATGGAGGCTGTGCCTACTGCCTCGGA Rgs6.896:(SEQ ID NO: 173) TGCTGTTGACAGTGAGCGCCAAGTGAAGATTGACCGGAAATAGTGAAGCCACAGATGTATTTCCGGTCAATCTTCACTTGTTGCCTACTGCCTCGGA Rock2.3898:(SEQ ID NO: 174) TGCTGTTGACAGTGAGCGCCAGATTCTATATGCCAATGAATAGTGAAGCCACAGATGTATTCATTGGCATATAGAATCTGATGCCTACTGCCTCGGA Rpa1.1620:(SEQ ID NO: 175) TGCTGTTGACAGTGAGCGCCCGCATGATCTTATCGGCAAATAGTGAAGCCACAGATGTATTTGCCGATAAGATCATGCGGTTGCCTACTGCCTCGGA Rpa3.561:(SEQ ID NO: 176) TGCTGTTGACAGTGAGCGCAAAAGTGATACTTCAATATATTAGTGAAGCCACAGATGTAATATATTGAAGTATCACTTTTATGCCTACTGCCTCGGA S100a9.148:(SEQ ID NO: 177) TGCTGTTGACAGTGAGCGCCAGACAAATGGTGGAAGCACATAGTGAAGCCACAGATGTATGTGCTTCCACCATTTGTCTGATGCCTACTGCCTCGGA Slc11a1.307:(SEQ ID NO: 178) TGCTGTTGACAGTGAGCGCAACATTGAGTCCGACCTTCAATAGTGAAGCCACAGATGTATTGAAGGTCGGACTCAATGTTTTGCCTACTGCCTCGGA Slc12a5.3582:(SEQ ID NO: 179) TGCTGTTGACAGTGAGCGAAACGAGGTCATCGTGAATAAATAGTGAAGCCACAGATGTATTTATTCACGATGACCTCGTTCTGCCTACTGCCTCGGA Slc15a3.1086:(SEQ ID NO: 180) TGCTGTTGACAGTGAGCGCCTGGTTCTATTGGAGCATCAATAGTGAAGCCACAGATGTATTGATGCTCCAATAGAACCAGTTGCCTACTGCCTCGGA Slc22a4.2223:(SEQ ID NO: 181) TGCTGTTGACAGTGAGCGCAAACGTATAAATGCTATCCAATAGTGAAGCCACAGATGTATTGGATAGCATTTATACGTTTATGCCTACTGCCTCGGA Slc6a13.1196:(SEQ ID NO: 182) TGCTGTTGACAGTGAGCGACTGGGACTAGATAGCCAGTTTTAGTGAAGCCACAGATGTAAAACTGGCTATCTAGTCCCAGGTGCCTACTGCCTCGGA Smpd2.592:(SEQ ID NO: 183) TGCTGTTGACAGTGAGCGACAGCATGTCTACAGTCTGAATTAGTGAAGCCACAGATGTAATTCAGACTGTAGACATGCTGGTGCCTACTGCCTCGGA Syt17.721:(SEQ ID NO: 184) TGCTGTTGACAGTGAGCGCCGAGGAGATCATGTCCAAGTATAGTGAAGCCACAGATGTATACTTGGACATGATCTCCTCGTTGCCTACTGCCTCGGA Tex14.3976:(SEQ ID NO: 185) TGCTGTTGACAGTGAGCGCCAGGAGCTACTTGATGAAATTTAGTGAAGCCACAGATGTAAATTTCATCAAGTAGCTCCTGATGCCTACTGCCTCGGA Thyn1.315:(SEQ ID NO: 186) TGCTGTTGACAGTGAGCGCAAGCAACTACTGGCTGATGAATAGTGAAGCCACAGATGTATTCATCAGCCAGTAGTTGCTTATGCCTACTGCCTCGGA Tnfsf12.1204:(SEQ ID NO: 187) TGCTGTTGACAGTGAGCGCAATGGATATTAAAGAGAATAATAGTGAAGCCACAGATGTATTATTCTCTTTAATATCCATTTTGCCTACTGCCTCGGA Trpm4.2813:(SEQ ID NO: 188) TGCTGTTGACAGTGAGCGACAAGATTGTCATAGTGAGCAATAGTGAAGCCACAGATGTATTGCTCACTATGACAATCTTGGTGCCTACTGCCTCGGA Vnn3.345:(SEQ ID NO: 189) TGCTGTTGACAGTGAGCGAACGCCAGAAGATGGAATCTATTAGTGAAGCCACAGATGTAATAGATTCCATCTTCTGGCGTCTGCCTACTGCCTCGGA Wnt10b.1717:(SEQ ID NO: 190) TGCTGTTGACAGTGAGCGCCTCGAATAGACTAAGATGAAATAGTGAAGCCACAGATGTATTTCATCTTAGTCTATTCGAGTTGCCTACTGCCTCGGA BRAF.3750:(SEQ ID NO: 191) TGCTGTTGACAGTGAGCGCTAGCATAATGACAATTATTTATAGTGAAGCCACAGATGTATAAATAATTGTCATTATGCTAATGCCTACTGCCTCGGA BRAF.3826:(SEQ ID NO: 192) TGCTGTTGACAGTGAGCGCCCCATTGTTTCTTCCAACTTATAGTGAAGCCACAGATGTATAAGTTGGAAGAAACAATGGGATGCCTACTGCCTCGGA BRAF.5053:(SEQ ID NO: 193) TGCTGTTGACAGTGAGCGCTAGGGTGATGTCTCACTTGAATAGTGAAGCCACAGATGTATTCAAGTGAGACATCACCCTATTGCCTACTGCCTCGGA Kit.1241:(SEQ ID NO: 194) TGCTGTTGACAGTGAGCGATTCCGTGACATTCAACGTTTATAGTGAAGCCACAGATGTATAAACGTTGAATGTCACGGAAGTGCCTACTGCCTCGGA Kit.2021:(SEQ ID NO: 195) TGCTGTTGACAGTGAGCGACACCCTGGTCATTACAGAATATAGTGAAGCCACAGATGTATATTCTGTAATGACCAGGGTGGTGCCTACTGCCTCGGA Kit.221:(SEQ ID NO: 196) TGCTGTTGACAGTGAGCGCCAGATGGACTTTCAAGACCTATAGTGAAGCCACAGATGTATAGGTCTTGAAAGTCCATCTGATGCCTACTGCCTCGGA Kit.4813:(SEQ ID NO: 197) TGCTGTTGACAGTGAGCGATTGGATATTCTTGAAAGTTTATAGTGAAGCCACAGATGTATAAACTTTCAAGAATATCCAAGTGCCTACTGCCTCGGA Lin28.2180:(SEQ ID NO: 198) TGCTGTTGACAGTGAGCGCAGCGTGATGGTTGATAGCTAATAGTGAAGCCACAGATGTATTAGCTATCAACCATCACGCTATGCCTACTGCCTCGGA Lin28.2186:(SEQ ID NO: 199) TGCTGTTGACAGTGAGCGAATGGTTGATAGCTAAAGGAAATAGTGAAGCCACAGATGTATTTCCTTTAGCTATCAACCATCTGCCTACTGCCTCGGA Lin28.2270:(SEQ ID NO: 200) TGCTGTTGACAGTGAGCGCAACGGGACAAATGCAATAGAATAGTGAAGCCACAGATGTATTCTATTGCATTTGTCCCGTTTTGCCTACTGCCTCGGA Lin28.2430:(SEQ ID NO: 201) TGCTGTTGACAGTGAGCGATGGCCTAGTTGTGTAAATATATAGTGAAGCCACAGATGTATATATTTACACAACTAGGCCACTGCCTACTGCCTCGGA Luciferase.1309:(SEQ ID NO: 202) TGCTGTTGACAGTGAGCGCCCGCCTGAAGTCTCTGATTAATAGTGAAGCCACAGATGTATTAATCAGAGACTTCAGGCGGTTGCCTACTGCCTCGGA Map2k1.1200:(SEQ ID NO: 203) TGCTGTTGACAGTGAGCGAGCCTCTCAGCTCATATGGAATTAGTGAAGCCACAGATGTAATTCCATATGAGCTGAGAGGCCTGCCTACTGCCTCGGA Map2k1.2337:(SEQ ID NO: 204) TGCTGTTGACAGTGAGCGCCAAGATGTTTATCAAATCTAATAGTGAAGCCACAGATGTATTAGATTTGATAAACATCTTGATGCCTACTGCCTCGGA Mn1.1403:(SEQ ID NO: 205) TGCTGTTGACAGTGAGCGCAACGGTACCCTAGACAACCAATAGTGAAGCCACAGATGTATTGGTTGTCTAGGGTACCGTTATGCCTACTGCCTCGGA Mn1.2545:(SEQ ID NO: 206) TGCTGTTGACAGTGAGCGCCAGCGCGGTTGCAGCCGGTAATAGTGAAGCCACAGATGTATTACCGGCTGCAACCGCGCTGTTGCCTACTGCCTCGGA Mn1.5760:(SEQ ID NO: 207) TGCTGTTGACAGTGAGCGCTTGGTTTAGCAGGAAGAATAATAGTGAAGCCACAGATGTATTATTCTTCCTGCTAAACCAAATGCCTACTGCCTCGGA Mn1.5864:(SEQ ID NO: 208) TGCTGTTGACAGTGAGCGCATCGCTATTGCACATGTATAATAGTGAAGCCACAGATGTATTATACATGTGCAATAGCGATTTGCCTACTGCCTCGGA Ptgs2.1082:(SEQ ID NO: 209) TGCTGTTGACAGTGAGCGCCAAGATAGTGATCGAAGACTATAGTGAAGCCACAGATGTATAGTCTTCGATCACTATCTTGATGCCTACTGCCTCGGA Ptgs2.2058:(SEQ ID NO: 210) TGCTGTTGACAGTGAGCGCCCGGTGTTTGTCCTTTAAATATAGTGAAGCCACAGATGTATATTTAAAGGACAAACACCGGATGCCTACTGCCTCGGA Ptgs2.284:(SEQ ID NO: 211) TGCTGTTGACAGTGAGCGAAAGAATCAAATTACTGCTGAATAGTGAAGCCACAGATGTATTCAGCAGTAATTTGATTCTTGTGCCTACTGCCTCGGA Ptgs2.3711:(SEQ ID NO: 212) TGCTGTTGACAGTGAGCGCAACGTTCATGGATAAATTCTATAGTGAAGCCACAGATGTATAGAATTTATCCATGAACGTTATGCCTACTGCCTCGGA Renilla.713:(SEQ ID NO: 213) TGCTGTTGACAGTGAGCGCAGGAATTATAATGCTTATCTATAGTGAAGCCACAGATGTATAGATAAGCATTATAATTCCTATGCCTACTGCCTCGGA Trp53.1224:(SEQ ID NO: 214) TGCTGTTGACAGTGAGCGCCCACTACAAGTACATGTGTAATAGTGAAGCCACAGATGTATTACACATGTACTTGTAGTGGATGCCTACTGCCTCGGA

1-29. (canceled)
 30. A method for treating chemotherapeutic-resistantacute myeloid leukemia in a subject in need thereof, wherein the subjectexhibits a known genotype associated with a chemotherapy-resistantleukemia, the method comprising: administering to the subject a HDAC3inhibitor, wherein HDAC3 expression is necessary for survival of thechemotherapy-resistant acute myeloid leukemia cell with said knowngenotype and is dispensable for the growth of non-transformedhematopoietic cells, so as to inhibit survival of acute myeloid leukemiacells in the subject, and thereby treat the chemotherapeutic-resistantacute myeloid leukemia in the subject.
 31. The method of claim 30,wherein the known genotype comprises a chromosomal rearrangementresulting in a MLL fusion protein, or AML1/ETO fusion protein.
 32. Themethod of claim 31, wherein the MLL or AML fusion protein is selectedfrom the group consisting of MLL/ENL, MLL/AF9, AML1/ETO9a.
 33. Themethod of claim 30, wherein the HDAC3 inhibitor is a hydroxamate. 34.The method of claim 30, wherein the HDAC3 inhibitor issuberanilohydroxamic acid (SAHA).
 35. A method for treatingchemotherapeutic-resistant acute myeloid leukemia in a subject in needthereof, wherein the subject exhibits a known genotype associated with achemotherapy-resistant leukemia, the method comprising: administering tothe subject SAHA, wherein SAHA inhibits HDAC3 and HDAC3 expression isnecessary for survival of the chemotherapy-resistant acute myeloidleukemia cell with said known genotype and is dispensable for the growthof non-transformed hematopoietic cells, so as to inhibit survival ofacute myeloid leukemia cells in the subject, and thereby treat thechemotherapeutic-resistant acute myeloid leukemia in the subject.